Tat-endophilin A1 was synthesized by cloning human endophilin A1 cDNA in a TA vector and Tat-1 expression vector. To visualize and compare the effects of endophilin A1 with and without the Tat-1 expression vector, the expression vector of endophilin A1 was constructed with a polyhistidine tag. Tat-SH3GL2 and Control-SH3GL2 plasmids were amplified and purified proteins were obtained as described previously [17 (link),18 (link)]. Purified proteins were confirmed by Western blot analysis using polyhistidine antibody (1:3000, Sigma, St. Louis, MO, USA) wherein the tagged protein was detected with chemiluminescent reagent as per the manufacturer’s instructions (Amersham, Franklin Lakes, NJ, USA).
Different concentrations of Tat-SH3GL2 and Control-SH3GL2 (0.5 to 5.0 μM) were incubated over a period of time (15 to 60 min) with 3 μM protein to observe the time- and concentration-dependent delivery of protein into HT22 cells. In addition, Tat-SH3GL2 was incubated for 60 h to elucidate the intracellular stability and degradation of Tat-SH3GL2 in HT22 cells. Intracellular delivery was confirmed by Western blot analysis using the specific antibody against the target protein as described previously [17 (link),18 (link)].
Different concentrations of Tat-SH3GL2 and Control-SH3GL2 (0.5 to 5.0 μM) were incubated over a period of time (15 to 60 min) with 3 μM protein to observe the time- and concentration-dependent delivery of protein into HT22 cells. In addition, Tat-SH3GL2 was incubated for 60 h to elucidate the intracellular stability and degradation of Tat-SH3GL2 in HT22 cells. Intracellular delivery was confirmed by Western blot analysis using the specific antibody against the target protein as described previously [17 (link),18 (link)].
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