Fig. 1.

Generation of a specific antibody against phospho-Ser316 (p-S316) of TTP. A The antibody specifically recognizes p-S316. HEK293T cells were overexpressed with wild-type, or S316A, or S318A, or S316,318A, or S52,178A of Flag-TTP. Cell extracts were isolated and with or without alkaline phosphatase treatment for western blotting analysis by using anti-TTP, anti-phospho-S316, and anti-Flag. B The antibody is specific for TTP not for Zfp36l1 and Zfp36l2. The amino acid sequences of NOT1-binding domain in TTP and Zfp36l1 (or Zfp36l2) were shown. HEK293T cells were transfected with pEGFP vector (V) or pEGFP- TTP family, TTP, Zfp36l1, or Zfp36l2, cell extracts were western blotting with anti-p-S316 and anti-GFP. C In vitro kinase assay. Recombinant GST-TTP(WT) or GST-TTP(S316A) was incubated with recombinant active kinases as indicated and then subjected to SDS-PAGE for western blotting by using anti-p-S316 and anti-TTP. D Knockdown of MK2 or RSK1 decreases Ser316 phosphorylation. HEK293T cells were transfected with siRNA of MK2 or RSK1 for 24 h and then overexpressed with Flag-TTP wild-type, S316A or S52, 178A. Cell extracts were harvested for western blotting analysis by anti-p-S316 and anti-Flag. All experiments were reproduced at least three times and the representative results were displayed