Inducible HSP70 modulated IL-1β-induced activation of TAK1-NF-κB cascades, but not of TAK1-MAPKs.

<p>(A) HeLa cells were transfected with pcDNA3.0 (−) or pcDNA3.0-Flag-HSP70 (+) for 48 h and then stimulated with IL-1β (10 ng/ml) for indicated times. Cell lysates were prepared and subjected to immunoblotting with anti-IκBα, anti-phospho-p38 (p-p38, Thr180/Tyr182), and anti-phospho-JNK (p-JNK, Thr183/Tyr185) antibodies, respectively, to reveal the activation of NF-κB and MAPKs. Total cellular TAK1, p38, and JNK protein levels in cell lysates were also determined by immunoblotting with specific antibody respectively. (B) HeLa cells were either non-transfected (control) or transfected with pRNA-U6.1/neo vector or pRNA-U6.1/neo-HSP70i construct. After 48 h transfection, cells were exposed to IL-1β (10 ng/ml) for 12 h and cell lysates were subjected to immunoblotting with anti-HSP70 antibody to determine the efficiency of RNAi, GAPDH was used as an internal control. (C) HeLa cells were transfected with either pRNA-U6.1/neo vector or pRNA-U6.1/neo-HSP70i construct. After 48 h transfection, cells were stimulated with IL-1β (10 ng/ml) for indicated times. immunoblotting with anti-IκBα, anti-phospho-p38 (p-p38, Thr180/Tyr182), and anti-phospho-JNK (p-JNK, Thr183/Tyr185) antibodies, respectively, to reveal the activation of NF-κB and MAPKs. Total cellular TAK1, p38, and JNK protein levels in cell lysates were also determined by immunoblotting with specific antibody respectively. Equal loading protein was confirmed by GAPDH.