Hsc70 prevented Rab1A degradation.

<p>(A) Hsc70-Rab1A interaction through chaperone activity. HT29 cells were subjected to serum depletion for 24 h, and then lysed. To verify the interaction between Hsc70 and Rab1A in a chaperone-dependent manner, anti-Hsc70 or anti-Rab1A immunoprecipitant was eluted with ATP, followed by elution with SDS sample buffer and immunoblotting in order to confirm the bait proteins. (B) Hsc70 knockdown decreased the level of Rab1A protein under stress conditions. HT29 cells transfected with <i>Hsc70</i> or <i>control</i> siRNA were subjected to serum depletion, 5-FU, or vehicle treatment for 24 h. Immunoblotting for endogenous Rab1A and Hsc70 proteins. β-actin was used as a loading control. (C) Hsc70 knockdown did not decrease <i>Rab1A</i> mRNA level. After knockdown of Hsc70 and in the control, <i>Rab1A</i> mRNA levels were determined by qPCR at 48 h post-transfection. (D) Hsc70 knockdown promoted the ubiquitination of Rab1A. After Hsc70 knockdown or control cells were lysed, immunoprecipitation (IP) with anti-Rab1A or anti-ubiquitin antibodies was performed, followed by immunoblotting with anti-ubiquitin or anti-Rab1A antibodies. (E) MG132 treatment inhibited Rab1A degradation. Hsc70 knockdown cells were subjected to serum depletion or 5-FU treatment, and then MG132 (10 µM) or vehicle was added for the last 8 h before sampling. SD, serum depletion. FU, 5-fluorouracil. Ub, ubiquitin, IgG LC, immunoglobulin light chain. Data (except in C) are representative of at least two separate experiments yielding similar results.