2.7 |. Protein kinase phosphorylation assay

Western blotting assay was carried out to determine the phosphorylation levels of ERKs, JNKs, and p38 kinase in according to the protocol of New England Biolabs (Beverly, MA). Briefly, the cultured JB6 cells in different groups were collected, homogenized, and separated by 10% SDS-PAGE. After electrophoresis, samples were transferred to PVDF membranes and incubated with diluted phospho-specific antibodies against phosphorylated sites of ERKs, JNKs, and p38 kinase (1:1000). Meanwhile, non-phospho-specific antibodies against ERKs, JNKs, and p38 kinase proteins provided in each assay kit were used to normalize the phosphorylation assay by using the same-transferred membrane blot.