HSP70 destabilized TAK1 at protein level via proteasome pathway.

<p>(A) HeLa cells were exposed to a mild heat shock (42°C for 30 min) followed by recovery at 37°C for indicated time periods. Total RNA was isolated and TAK1 mRNA was detected by RT-PCR using primers indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050059#s2" target="_blank">Material and Methods</a>. β-actin was used as control. (B) HeLa cells were transfected with pcDNA3.0-Flag-HSP70 (+) or pcDNA3.0 (−). After 48 h, cells were treated with 1 µg/ml cycloheximide (CHX) and harvested at indicated time points. TAK1 protein levels were determined by Western blotting, GAPDH was used as loading control, and antibody against Flag-tag was used to show the overexpression of HSP70. (C) HeLa cells were transfected with pcDNA3.0-Flag-HSP70 (+) or pcDNA3.0 (−). After 48 h, cells were treated with a proteasome inhibitor MG-132 at indicated concentrations for 6 h. Cells were collected and subjected to immunoblot analysis with TAK1 antibody. Equal loading protein was confirmed by immunoblotting with anti-GAPDH antibody.