Fig. 3.

Ser316 is phosphorylated in LPS-treated RAW264.7 cells. A The kinetic analysis of Ser316 phosphorylation. RAW264.7 cells were treated with 100 ng/ml of LPS for indicated time intervals. Whole cell extracts were isolated for western blotting analysis as indicated. B Whole cell extracts from control and LPS-treated RAW264.7 cells were isolated and western blotting analysis with antibodies as indicated. C RAW264.7 cells were pretreated with 2 μM of MK2 (PF3644022) or 25 μM of RSK1(BI-D1870) inhibitors for 0.5 h and followed by LPS stimulation for 1 h. Whole cell extracts were isolated and western blotting analysis with antibodies as indicated, and RNA was isolated for RT-qPCR with control β-actin and TNFα primers. **P<0.01. D Solid phase kinase assay. GST-TTP bound on glutathione –sepharose was incubated with or without LPS-treated cell lysates. After extensive washes, kinase assays were performed. RSK1 and MK2 were included as positive controls. The samples were separated on SDS-PAGE and western blotting with anti-p-S316 and then ponceau S staining