The functional analysis of Ser316 phosphorylation in LPS-stimulated RAW264.7 cells. A Kinetics of TNFα mRNA expression in LPS-treated wild-type and TTP KO cells. B TNFα mRNA stability analysis. Wild-type and TTP KO RAW264.7 cells were treated with 100 ng/ml of LPS for 30 min and then adding 10 μg/ml of actinomycin D to block transcription for 15, 30, and 45 min. Total RNAs were isolated to perform RT-qPCR with TNFα and β-actin primers and the percent of RNA remaining was shown. C TTP rescue analysis. TTP KO cells were transfected with GFP vector, or GFP-TTP(S316A), or GFP-TTP(S316D). After 24 h, the cells were treated with 100 ng/ml of LPS for 0.5, 1, and 2 h, and RNA was isolated for qPCR analysis with TNFα and β-actin primers. All experiments were performed independently at least three times, and error bars represent mean ± S.D.