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Delivery of proteins, peptides or cellphone-impermeable small molecules in living cells by incubation with the endosomolytic reagent dfTAT
Macromolecular delivery strategies typically use the endocytic route as the route of cellular entry. However, endosomal entrapment limits the efficiency in which macromolecules can penetrate a cytosolic space of the cells. Recently we circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We created a fluorescently labeled dimer of the prototypical cell penetrating peptide (CPP) TAT dfTAT, which penetrates living cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT was achieved in several cell lines, including primary cells. In addition, the delivery does not noticeably affect cell viability, proliferation, or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in DfTa T synthesis and cell delivery. The manuscript describes how to control the amount of protein supplied to the cytosolic space of cells by controlling the amount of protein administered extracellularly. Finally, the respective limitations of this new technology and steps involved in validation delivery are discussed. The protocols described should be used for cell-based assays as well as for the ex vivo manipulation and reprogramming cells can be extremely useful.
The administration of proteins, peptides or cell-impermeable small molecules into living cells is common in many biological or biotechnological applications (cellular imaging, functional assays, reprogramming, Etc.) 1-4 desirable. Many delivery approaches have been reported, including microinjection, electroporation, or the use of vehicles (e.g. E.g. cell penetrating peptides such as TAT, lipids) 5-7. Each technique usually has specific advantages and disadvantages that might make these approaches appropriate for certain applications but not for others. Common problems include poor delivery efficiencies and / or lack of control over how much material 8,9 delivered toxicity or adverse physiological effects 10,11, lack of time control, delivery in a few cells, but not in a total population (e.g. B. Microinjection) 12, and complex chemical conjugation or formulation systems 11.
s = "jove_content"> Recently we have developed a new delivery strategy that bypasses these restrictions. This strategy relies on a peptide called dfTAT (Dimer Fluorescence TAT) 13. dfTAT is derived from the well known cell penetrating peptide (CPP) TAT. dfTAT contains two disulfide-bound copies of TAT labeled with the fluorophore tetramethylrhodamine. Despite their similarities, TAT and dfTAT differ significantly in activity. TAT is typically internalized into cells by endocytosis. However, the CPP mostly remains trapped within endosomes (this usually lead to a punctiform distribution of the peptide in cells when examined by fluorescence microscopy). Like TAT, dfTAT is efficiently endocytosed by cells. However, dfTAT does not mean staying trapped in endosomes. Instead, it mediates endosomal leakage in a way that is very efficient. The endosomolytic activity of dfTAT can then be used to deliver macromolecules through a simple incubation assay.ntent "> The current understanding of the delivery process is as follows. dfTAT induces macropinocytosis. As a result, cells incubated with dfTAT take soluble proteins, peptides or small molecules (molecules of interest, MOI) present in media through liquid-phase endocytosis (see Illustration 1). The interactions between dfTAT and MOI are not required as long as both entities traffic together in the endocytic. As dfTAT reaches a certain threshold in the lumen endocytic organelles, expressing their endosomal leakage activity (the molecular details remain are fully characterized). The contents of the lumen of leaky organelles, and therefore the MOI, is then released into the cells. This approach is therefore very convenient as it does not require conjugation or preparation schemes with MOI. Furthermore, since dfTAT does not directly modify an MOI, it should also not interfere with MOIs function when intracellular delivery is achieved. In addition, the concentration used by ddfTAT delivery is independent of that used by the Ministry of Interior in media. For example, dfTAT concentration can be kept constant between experiments to ensure reproducible efficiencies in endosomal leakage. In contrast, the concentration of MOI in media can be gradually changed to achieve desired levels of MOI released in cytosol.
The high efficiency achieved in the endosomal leak dfTAT is remarkably harmless to many of the cells that have been tested so far. This is a surprise because endocytic organelles are an important part of cells and one would expect that the dramatic leakage mediated by dfTAT would be accompanied by harmful cellular responses. However, treated cells multiply at the same rate as untreated cells and do not display any significant changes in their transcriptome. In addition, they can be repeated in minutes with reproducible delivery efficiencies, indicating that the cells are either tolerating or recovering from the delivery process without losing their ability to endocytose or endosomal leakage. Subtle cellular responses might take place during dfTAT delivery and the molecular details of what those responses might remain to be explored. However, due to the high economy, convenience of the protocols, and the lack of toxicity, this delivery approach immediately proves useful in many cell-based applications. The protocols given here work to make this technology accessible to the research community.
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1. SPPS: CK (TMR) TATG (fTAT) synthesis
Note: dfTAT is produced in two steps: synthesis of the monomer fTAT by solid phase peptide synthesis followed by a disulfide bond to form dimerization dfTAT.
- Swell 500 mg of Rink Amide MBHA resin in dimethylformamide (DMF) in a standard 50 ml SPPS jar for 1 hour.
- Introduce Fmoc cleavage by incubating the Fmoc protected resin in a 20% piperidine solution (20% piperidine in DMF, 10 ml (0.30 mmol). Perform deprotection steps twice (1 x 5 min and 1 x 15 min) with a Washing step with DMF (the washing step involves rinsing the resin several times with a total volume of about 150 ml DMF and dissolving the solvent by vacuum filtration) in between reactions.
- Synthesize CK (TMR) RKKRRQRRRG (fTAT) on the Rink Amide MBHA Resin. Use the following Fmoc-protected amino acids Fmoc-Lys (Mtt) -OH (only at the N-terminus), Fmoc-Lys (Boc) -OH, Fmoc-Gly-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH and Fmoc-Cys (Trt) -OH. The reaction With N 2 (20 psi) to stir the reaction. Perform all reactions at RT.
- Perform each amino acid coupling reaction for 4 hours. For each coupling use the Fmoc protected amino acid (1,2 mmol), N, N, N ', N' -Tetramethyl- O- (1-benzotriazol-1-yl) Dissolved uronium hexafluorophosphate (HBTU) (0.44 g, 1.1 mmol) and diisopropylethylamine (DIEA) (0.51 ml, 3.0 mmol) in DMF. After 4 hours of coupling, wash the resin with DMF and perform the Fmoc deprotection step as described in step 1.2.
- Repeat until the linear peptide chain fTAT (Linear Peptide Chain: CKRKKRRQRRRG no TMR) is synthesized.
- Then wash resin thoroughly with first DMF and after that with dichloromethane (DCM). With a total volume of approximately 150 ml DMF or DCM, rinse the resin several times and remove the solvent by vacuum filtration.
- Use MALDI-TOF to confirm that the obtained peptide has the correct molecular weight.
- A matrix mixture consists of adding 1 mg of matrix to a solution of 50 μl of 0.01% TFA in water solution and 50 μl of acetonitrile.
- To confirm linear peptide chain mass, use α-cyano-4-hydroxycinnamic acid. Perform analytical HPLC on the crude peptide and collect 50 μl from the top of the pure peak.
- To prepare the sample to plate on the MALDI plate: Add 8 μl of the peptide to 2 μl. l the matrix solution and dry on the MALDI plate or on a 37 ° C hot plate in the air.
Note: Arginine is known to be a difficult amino acid pair in SPPS. Successful pairing by running a Kaiser test 14(e.g. Ninhydrin test) established. This test enables the detection of free amines that could remain uncoupled on the resin. If a blue color is found (evidence of the presence of free amines), coupling sTEPs are repeated.
- CK (-NH-TMR) TATG (fTAT), cleave the Mtt protecting group at the & egr; -Amino group of Lys on CK (-NH-Mtt) TATG with a solution of 1% trifluoroacetic acid (TFA) and 2% triisopropylsilane together (TIS) in DCM.
- Removal of the MTT protecting group would result in the appearance of a yellow color, incubate the resin with 20 ml of the above solution for 5 min, repeat until no yellow color is observed. In between the resin with DCM and DMF. In addition, since TIS is a scavenger that the MTT would intercept as soon as no yellow color appears in the solution in the presence of MTT.
- Adding an additional solution with 1% TFA in DCM (no TIS) to the resin to insure no more MTT is removed and the solution remains clear.
- Dissolve the three components: carboxytetramethylrhodamine (TMR), HBTU and DIEA (4, 3.9 and 10 equivalents in terms of the amount of resin (in mg)) in DMF and this mixture to the resin and the reaction O / N with dry N 2, to create unrest.
- After Fmoc deprotection and amino acid conjugation, coat the resin with DCM and allow to dry. For complete cleavage of the peptide from the resin, a solution of 92.5% TFA, 2.5% H 2 O, 2.5% TIS, 2.5% ethanedithiol (EDT) (total volume = 4 ml) to achieve the peptidyl resin for 3 h at RT to remove the protective groups and cleavage from the resin.
- Precipitation of the crude peptide products with cold anhydrous ethyl ether (Et 2 O) by draining the solution from stage 1.6, 40 ml of Et 2 O, turn this down to 4 ° C and 4,000 × g for 20 - 25 min.Repeat this step to wash the precipitate with cold anhydrous Et 2 O enable
- Resuspend the precipitate in water (5 ml, or until precipitate is completely dissolved) and lyophilize. Then resuspend in 0.1% aqueous TFA / acetonitrile products obtained.
- An HPLC analysis with a C18 analytical column (5 µm, 4 x 150 mm) to analyze each peptide. Use a flow rate of 1 ml / min and detection at 214 nm and 550 nm.
- Apply semi-preparative HPLC on a C18 10 x 250 mm column for peptide purification. Use a flow rate of 4 ml / min and detection at 214 nm and 550 nm. For all runs use linear gradients of 0.1% aqueous TFA (Solvent A) and 90% acetonitrile, 9.9% water and 0.1% TFA (Solvent B).
- Confirm the correct identity of the peptides by MALDI-TOF according to the manufacturer's protocol: fTAT, expected mass: 2,041.17, observed mass: 2,040.66. DEAC-K9, expected mass: 1,412.97, observed mass: 1,415.59. Use an α-cyano-4-hydroxycinnamic acid matrix for the MALDI-TOF.
2. Oxidation: dfTAT creation
- Aerate phosphate buffered saline (PBS), pH 7.4 for 1 hour (at least 5 ml for the reaction below).
- Solve fTAT (0.3 mg, 1.5 x 10 -4 mmol) in aerATED PBS pH 7.4 (5 ml). Make sure the pH is between 7.0 to 7.5 after adding the peptide. If not, add sodium hydroxide (1 M, in small increments of 1-5 µl) to bring the pH back to 7.4.
- Note: Oxygen dissolved in PBS acts to oxidize the thiol groups on fTAT and form a disulfide bond.
- Nutated the reaction O / N to allow it to react to completion (100% yield, based on HPLC analysis). Purify the product using reverse phase HPLC and mass spectrometry (MALDI-TOF) analyzed as in step 1.9. Expected mass: 4,080.34, observed mass: 4,084.21.
- Lyophilize pure dfTAT (0.71 x10 -4 mmol) and resuspend in 200 µl of water (concentration of pure dfTAT = 356.3 µM).
3. Measure dfTAT concentration
- Resuspend an aliquot of the purified dfTAT (typically 1 µl depending on the amount of peptide purified) in a 149 µl 50 mM TCEP solution.
- Note: In this step dfTAT is reduced in its mo against fTAT to quench the absorption, which is due to the close proximity of the TMR fluorophore in dfTAT (the extinction coefficient ε of TMR in dfTAT is reduced compared to the ε of TMR in reduced fTAT) too eliminate.
- The sample will react for approximately 20 min (analytical HPLC can be used to confirm the formation of fTAT).
- Add all of the solution to the quartz cuvette and measure the absorbance at 556 nm.
- Using Beer's law (A = εcl: ε = 91,500 M. -1 cm -1) to calculate the concentration of fTAT, determine the concentration of dfTAT, and divide [fTAT] by two.
4. Cellular delivery experiments
- Plant seeds, the cells (HeLa, HDF, Etc.) In a bowl at a confluence of 80-90% (e.g. 8-well or 24-well dish). 90% confluence in a 37 ° C - cells grow in a suitable medium until 80 (e.g. DMEM supplemented with 10% FBS and Pen / Strep) using a humidified atmosphere 5% CO 2.
- The cells are replenished three times (3x) with PBS (by adding 200 μl of PBS and then removing three times).
- Make a 5 µM working concentration of dfTAT by diluting a strain of dfTAT (in water) in NRL-15 media (for an 8 well dispensing the total volume should be 200 µl). A concentration of 5 µM dfTAT results in efficient delivery (high cytosolic delivery in over 90% of the cells present in a dish) in most up-to-date (Illustration 3) cell types tested. However, lower or higher concentrations may be more sufficient for cell types with a high or low propensity to invade.
NOTE ON MEDIA: NRL-15 is used for the delivery of dfTAT as it is not cysteine, which could reduce the disulfide bond in dfTAT. However, our data show that both regular L-15 (with cysteine) and DMEM can be used as delivery media. L-15 contains the reducing amino acid cysteine, but cysteine presumab is oxidized in the media to form cystine (DMEM formulated with cystine). dfTAT therefore remains intact in these media and supply plants with the same efficiency as that with nrL15.
- Incubate cells with dfTAT (5 µM) with or without load (e.g. B. EGFP (10 µM)) and hold at 37 ° C for 1 hour (incubation time reduced, but dfTAT typically requires about 30 to 45 min to induce the endosomal leak).
- In L-15 cells, wash with heparin (1 mg / ml) (3 washes are recommended) to remove dfTAT which is bound to the plasma membrane of the cells.
- Cells with cell-impermeable nuclear stain, incubation z. B. Sytox Blue, Sytox Green (2 µM in NRL-15) to determine if the plasma membrane of the cells is compromised according to the manufacturer's protocol (dead cells that are not stained while living cells are stained).
- Image cells with a fluorescence microscope (100x oil immersion or 20x objective). Image dfTAT with an RFP filter (Ex = 560 ± 20 nm / Em = 630 ± 35 nm).
Note: Successful delivery results in a diffuse fluorescence of dfTAT throughout the cell (assessment of the release of protein or peptide of interest will depend on application). Staining of nucleoli by fTAT (the reduced product of dfTAT on cytosolic input) can be used as an indication that the detected fluorescence is intracellular. fTAT is broken down within a few hours. At this point in time, the fluorescence of the degradation fragments is displayed as punctiform. This should not be confused for the punctiform distribution that can also be seen when dfTAT remains trapped inside endosomes (this can happen if dfTAT is present at too low a concentration).
5. Controlling concentration of MOI delivery
- Identify the "optimal delivery" concentration required to achieve efficient cytosolic dfTAT release in the cell type used. Perform fluorescence microscopy on cells incubated with increasing concentrations of dfTAT.The optimal dfTAT concentration is defined as the minimum concentration that leads to quenching cytosolic "diffuse" TMR fluorescence in approximately 100% of the cells in a culture.
- The concentration of the MOI used in the coincubation protocol varies while the concentration of dfTAT is constant (e.g. using optimal delivery concentration dfTAT'S). Changing the incubation time to less than 1 hour could also be an option to vary the amount MOI to be delivered.
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To assess the difference between fTAT and dfTAT, HeLa cells are incubated for 1 hour with each peptide to determine the difference in their cellular location. The internalization of the two CPPs was examined by means of fluorescence microscopy. 2 shows that fTAT (20 uM) localized in a punctiform distribution. This distribution is consistent with the peptide including endosomes still in the device. In contrast, the fluorescence signal dfTAT (5 µM) shows a homogeneous distribution in the cytosol and nucleus. 3 shows that The cytosolic distribution of dfTAT is observed in a number of different cell lines including COLO 316, NIH 3T3, HaCaT, the difficult to transfect Neuro-2a, MCH58, the primary cell line, HDF, DRG-F11 and NCL-H1299. The 20X images shows that a very high percentage (> 80%) in a plate show a cytosolic distribution of dfTAT with no cellular toxicity (no Sytox blue core staining).
To determine whether dfTAT-mediated endosomal leakage delivers large proteins into the cytosol of cells. EGFP (26 kDa) is chosen as the model protein. This is because their fluorescence can be used to detect its delivery into the cells by observing the localization of the green fluorescence (if correctly folded). To use this assay, EGFP and DTAT were incubated with the cells for 1 hour, as in FIG 4 shown EGFP displayed a cytosolic and nuclear green fluorescent distribution similar to what was observed for dfTAT in greater than 90% of cells with no observable toxicity.
Figure 1. Schematic overview of the principle of dfTAT mediated molecular delivery. Left to right. Scheme shows dfTAT cellular delivery along with a cell impermeable cargo. First dfTAT induces endocytosis, which in the uptak yielded the dfTAT along with the charge in endocytic vesicles. In the second step, dfTAT escapes from the endocytic vesicle, which leads to the release of dfTAT and the cell-impermeable molecule in the cytosol of the cells. The cytosol of mammalian cells is a reductive environment, so in the cytosol dfTAT is reduced in its monomer against fTAT. Please click here to see a larger version of this figure.
Figure 2. Fluorescence and bright field images of HeLa cells with 20 µM fTAT (left image) and 5 µM fTAT. M dfTAT (right) with a 100x objective. FTAT monochrome images show cells that have a fluorescent dot-like distribution, while dfTAT images shows; Cells displaying a homogeneous cytosolic and nuclear fluorescenc e distribution. Scale:. 10 µ m Please click here to see a larger version of this figure.
Figure 3. Efficient delivery of dfTAT into living cells has been achieved in several cell lines. The cell lines tested were the following: HeLa, NIH 3T3, COLO 316 and HaCaT, Neuro-2a, MCH58, HDF, DRG-F11 and NCL H1299. Cells were incubated with 5 µM dfTAT for 1 hour followed by a wash according to protocol and imaged. The detected fluorescence signal was in the cytosol and nucleus of the cells (top: 20x objective, bottom plate: 100X objective). The cell-opaque nuclear stain SYTOX Blue was used to assess cell viability after dfTAT treatment. Scale bar, 20x objective: 50 μm m; 100X target: 10 µ m.jove.com/files/ftp_upload/53175/53175fig3large.jpg "target =" _ blank "> Please click here to see a larger version of this figure.
Figure 4. dfTAT Delivers Intact EGFP in Different Cell Lines. (A) HeLa (top panel) and NIH 3T3 (bottom panel) cells were incubated with EGFP (10 µM) and dfTAT (5 µM) for 1 h and the following step was performed, according to the protocol. Images show a homogeneous cytosolic fluorescence distribution of EGFP in both cell lines. Scale bar, 10 µ m. (B) HeLa and Grund HDF cells were incubated with EGFP (10 µM) and dfTAT (5 µM) according to the protocol. 20X images show a homogeneous cytosolic fluorescence distribution of EGFP and dfTAT in HeLa and Grund HDF cells. Overlay (pseudo: white) show presence of dfTAT (pseudo: purple), EGFP (pseudocoloder: green) in the cytosolic space of both HeLa and HDF cells. Sytox blue (blue) used on donkeys for cell viability. Scale bar :. 50 µ m Please click here to see a larger version of this figure.
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The dfTAT used for delivery should not be excessively confluent cells (> 90% confluence) as this could affect the delivery efficiency. The cells should also be healthy: dead cells in the culture can be apoptotic fragments with the dfTAT (e.g. DNA from degraded nuclei) interact, dissolve. This, in turn, can interfere with delivery efficiency and the quality of the imaging. Cells should be washed thoroughly to remove FBS before dfTAT. BSA present in FBS can bind dfTAT, and this can lower dfTAT delivery efficiency. Washes should be carried out with caution, however, as excessive force can cause adherent cells to loosen from the culture dish or to a load that results in the lower endocytic uptake. When delivering a protein / peptide with dfTAT the protein / peptide stock sample should be sufficient concentrated to avoid undue dilution of the NRL-15 media during incubation: e.g. adding 5 to 10 µg l of the sample to 200 μl NRL-15 is recommended.
Fluorescently labeled cell-penetrating peptides can indicate a light-inducible membrane disruptive activity. 15 This is the case for dfTAT when high intensity light doses are used. It can spread through rupture of the intracellular organelles (e.g. Endosomes, mitochondria), cell surfaces blebbing and cell death manifest. To minimize these effects, care should be taken to keep light exposure to a minimum (standard confocal or epifluorescence conditions are usually tolerated as required for imaging).
In the delivery of MOI, like proteins, one gets to the problem that every macromolecule is unique and that consequently every delivery experiment could require fine-tuning (more so than in the case of DNA transfection, where the molecules delivered are always a negative charged faced polymer made A, T, G and C). Problem solving is therefore important and some issues should be considered. First, proteins with very low pIs can electrostatically bind to dfTAT and inactivate them. In contrast, proteins with very high PIs dfTAT could compete for binding with negatively charged glycosaminoglycans on the cell surface. This could then reduce endocytosis and lessen the uptake below endosomolytic thresholds. In both cases, a possible solution to this problem is increasing the concentration of dfTAT.
As one would expect, due to the presence of the cellular proteases (e.g. cathepsins 16) along the endocytic, degradation of the MOI during delivery is a concern. While dfTAT can deliver intact proteins, the amount of protein that is completely or partially broken down during the delivery process has not been demonstrated. This is again an issue that is MOI-dependent and that should be monitored depending on the intended application.
dfTAT is a highly efficient delivery agent. However, the molecular basis for dfTAT activities remaindered by other people was unclear. In particular, the structural or chemical properties required to achieve endosomal escape have not been identified. Therefore, it is currently not possible to predict how much the dfTAT structure will be modified without changing delivery efficiencies. We have already established that the present disulfide bond in dfTAT can be replaced by an irreducible linker without deleterious effects. Additional structure-activity relationships are currently being established.
Our data suggest that it is possible to reduce the incubation time of the peptide to less than 1 hour (as low as 5 minutes was done). However, the release of dfTAT within the cells is not observed in a large population of cells until approximately 15-30 min. This suggests that short incubation times may be sufficient for dfTAT endocytosis or cellular uptake. However, for endosomal maturation for dfTAT to escape from the endocytic time is required.
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|nrL15 L-15 (-) cysteines||Hyclone||Special order|
|Fmoc-protected Amino acids||Novabiochem|
|dfTAT||Samples will be provided upon request (contact: [email protected])|
|Inverted epifluorescence microscope||Olympus||model IXB1||equipped with a heating stage maintained at 37 ° C and with a Rolera-MGI Plus back-illuminated electron-multiplying charge-coupled device (EMCCD) camera (Qimaging).|
|37 ° C humidified, 5% CO2 incubator|
|Peptide synthesizer or vessel to preform manual peptide synthesis|
DOWNLOAD MATERIALS LIST
- Didenko, V. V., Ngo, H., Baskin, D. S. Polyethyleneimine as a transmembrane carrier of fluorescently labeled proteins and antibodies.Anal Biochem. 344, 168-173 (2005).
- Takeuchi, T., et al.Direct and Rapid Cytosolic Delivery Using Cell-Penetrating Peptides Mediated by Pyrenebutyrate.ACS Chem Biol. 1, 299-303 (2006).
- Morris, M. C., Depollier, J., Mery, J., Heitz, F., Divita, G. A peptide carrier for the delivery of biologically active proteins into mammalian cells.Nat Biotech. 19, 1173-1176 (2001).
- Zhang, H., et al.Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors.Biomaterials. 33, 5047-5055 (2012).
- Torchilin, V. Intracellular delivery of protein and peptide therapeutics.Drug Discov Today: Technol. 5, e95-e103 (2005).
- Chakravarty, P., Qian, W., El-Sayed, M. A., Prausnitz, M. R. Delivery of molecules into cells using carbon nanoparticles activated by femtosecond laser pulses.Nature nanotechnol. 5, 607-611 (2010).
- Kaczmarczyk, S.J., Sitaraman, K., Young, H.A., Hughes, S.H., Chatterjee, D.K. Protein delivery using engineered virus-like particles.Proc Natl Acad Sci U S A. 108, 16998-17003 (2011).
- Pan, C., Lu, B., Chen, H., Bishop, C. Reprogramming human fibroblasts using HIV-1 TAT recombinant proteins OCT4, SOX2, KLF4 and c-MYC.Mol Biol Rep. 37, 2117-2124 (2010).
- Fittipaldi, A., et al.Cell Membrane Lipid Rafts Mediate Caveolar Endocytosis of HIV-1 Tat Fusion Proteins.J Biol Chem. 278, 34141-34149 (2003).
- Lee, Y. J., Erazo-Oliveras, A., Pellois, J. P. Delivery of macromolecules into live cells by simple co-incubation with a peptide.Chembiochem. 11, 325-330 (2010).
- Angeles-Boza, A. M., Erazo-Oliveras, A., Lee, Y. J., Pellois, J. P. Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans.Bioconjugate chem. 21, 2164-2167 (2010).
- Walev, I., et al.Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O.Proc Natl Acad Sci U S A. 98, 3185-3190 (2001).
- Erazo-Oliveras, A., et al.Protein delivery into live cells by incubation with an endosomolytic agent.Nat methods. 11, 861-867 (2014).
- Kaiser, E., Colescott, R. L., Bossinger, C. D., Cook, P. I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides.Anal Biochem. 34, 595-598 (1970).
- Muthukrishnan, N., Johnson, G.A., Lim, J., Simanek, E. E., Pellois, J. P. TAT-mediated photochemical internalization results in cell killing by causing the release of calcium into the cytosol of cells.Biochim biophys acta. 1820, 1734-1743 (2012).
- Lautwein, A., et al.Human B lymphoblastoid cells contain distinct patterns of cathepsin activity in endocytic compartments and regulate MHC class II transport in a cathepsin S-independent manner.J Leukoc Biol. 75, 844-855 (2004).
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