Western blot assay was performed as described by Sale et al [19 (link)]. Total proteins were extracted from cells using RIPA lysate (R0010, Solarbio) containing phenylmethylsulfonyl fluoride protease inhibitor (PMSF, P0100, Solarbio). Then, protein concentration was detected with a BCA kit (PC0020, Solarbio). After separated by 5-10% SDS-PAGE, transferred to the PVDF membrane, blocked by nonfat milk (A600669, Sangon Biotech, China), proteins on the membrane were incubated overnight at 4°C with the primary antibodies: iNOS (1: 1000, A0312, Abclonal, China), COX-2 (1: 2000, A1253, Abclonal), MMP-1 (1: 2000, A1191, Abclonal and 1: 1000, DF6325, Affinity), MMP-3 (1: 2000, A11418, Abclonal), MMP-13 (1: 2000, A11148, Abclonal and 1: 1000, AF5355, Affinity), p-ERK1/2 (1: 1000, AF1015, Affinity, China), ERK1/2 (1: 1000, AF0155, Affinity), p-p38 (1: 500, AF4001, Affinity), p38 (1: 500, AF6456, Affinity), p-JNK (1: 1000, AF3318, Affinity), JNK (1: 500, AF6318, Affinity), p-p65 (1: 1000, AF2006, Affinity), p65 (1: 1000, AF5006, Affinity), and GAPDH (1: 10,000, 60,004-1-Ig, Proteintech, China). Next, the membrane was incubated with appropriate secondary antibody. Finally, membranes were detected by Western electrogenerated chemiluminescence (ECL) Substrate (D1010, Solarbio) and the images were analyzed by Gel-pro analyzer software (Media Cybernetics, CA, USA).
Free full text: Click here
