Figure 3.

Involvement of JNK and IKK in regulating the phosphorylation of IRS1. Reverse transfection of suspended HepG2 cells was performed with scramble siRNA (negative control) or siRNA of PKR (siPKR) for 24 h, and the transfected cells were cultured in regular medium for another 24 h (A). Cells were harvested, and Western blot analysis was performed to detect the total and phosphorylated levels of JNK and IKK (A). Reverse transfection of suspended HepG2 cells was performed with scrambled siRNA (control) or siRNAs of JNK1 and JNK2 together or siRNA of IKK for 24 h, and the transfected cells were cultured in regular media for another 24 h. Next, the forward transfection of empty vector pCMV6-XL5 (pCMV6) or plasmid containing PKR cDNA sequence (pCMV6-hPKR) was performed, followed by insulin treatment (0.5 nM) for 15 min (B). After treatment, cells were then harvested, and Western blot analysis was performed to detect the protein level of IKK and JNK and total and phosphorylated levels of PKR and IRS1 (B).