P38 antibody

For total lysates, cells were directly lysed in 2 × SDS sample buffer (Invitrogen). Lysates were sonicated to shear DNA, and equivalent lysate levels were loaded onto SDS PAGE gel, blotted to nitrocellulose membrane, and probed with antibodies to TTP, phospho p38 (P-P38), phospho-MK2, MK2, and β-actin. Affinity-purified TTP rabbit polyclonal antibody was custom-made with Genescript against the C-terminal end of TTP PRRLPIFNRISVSE and was used previously (27 (link)). The specificity of the antibody was tested in western blots from transfected and/or LPS-induced cells and with pre-immune serum. P38 antibody was purchased from Santa Cruz Biotechnology; anti-phospho-p38 (Thr180/Tyr182) was purchased from Millipore/Merck; β-actin, MK2, and P-MK2 (Thr334) antibodies were purchased from Cell Signaling. For calf intestinal phosphatase (CIP) (Promega) treatment, cells were lysed in 1 × CIP buffer supplemented with 0.5% NP40. Typically, 100 μl of lysate was treated with 10 units of CIP for 30 min at 37°C.

A549 cells were purchased from American Type Culture Collection (ATCC) and cultured in DMEM (Invitrogen), supplemented with 10% FBS and antibiotics (Invitrogen). Dexamethasone sodium phosphate 4 mg/ml was from Simplist/USA. Dilutions were made in cell culture medium to reach a final concentration of 100 nM treatments. For western blots, cells were directly lysed in 1 × SDS sample buffer (Invitrogen). Lysates were sonicated to shear DNA, and equivalent lysate levels were loaded onto SDS PAGE gel, blotted to nitrocellulose membrane, and probed with antibodies to ZFP36, and GAPDH. Affinity-purified ZFP36 rabbit polyclonal antibody was custom-made with Genescript and was used previously^{5 (link)}. GAPDH, Phspho-P38 antibody was purchased from Cell Signaling. P38 antibody was purchased from Santa Cruz. IL1 alpha was from R&D systems, and was used to treat the cells at a 3ng/ml concentration.