297692.fig.003
Figure 3: Protein transduction into 293T cells and interaction of recombinant TN122 with the NHR2 domain. (a) Protein transduction of 1 µM TN122-eGFP into 293T cells in the presence or absence of 100 μM chloroquine and addition of 10 μM heparin. After 24 h, the cells were treated with trypsin (10 min, 37°C) and analyzed for the percentage of eGFP positive cells by flow cytometry. (b) Incubation of 1 μM TN122 for various times in serum-containing medium at 37°C and analysis of the stability of the protein by western blotting. (c) TAT-mediated protein transduction. Incubation of 293T cells with either 10 μM ΔT-NGFP or 2 μM TN122-eGFP in the presence of 100 μM chloroquine for 4 h. Thereafter trypsin treatment and western blot analysis of the myc-tagged proteins. (d) Influence of chloroquine on protein transduction. The 293T cells were incubated with 1 μM TN122 in the presence or absence of 20 μM chloroquine for the indicated time, treated with trypsin, and analyzed for the myc-tagged TN122 protein by western blotting. (e) Concentration dependency of protein transduction. Incubation of 293T cells with 2 μM (light gray) or 6 μM (dark gray) TN122-eGFP for 3 h and trypsin treatment followed by flow cytometry. The content of eGFP positive cells is indicated. (f) Binding of the recombinant cell-penetrating NHR2 polypeptide to ETO protein sequences. Protein transduction of 2 × 5 μM TN122 (at 0 and 24 h) in the presence of 20 μM chloroquine into 293T cells that stably express the NHR2-containing polypeptide NC128. The cells were treated with trypsin 14 hours after the last addition of the protein. The Flag-tagged NC128 was immunoprecipitated and the co-precipitated TN122 detected by western blotting. The blot was also stained for eGFP and actin to verify the specificity of the interaction.