Df6325

Total protein was extracted with RIPA lysis buffer from the indicated cells, and the concentration was measured with the BCA Protein Assay Kit (P0012, Beyotime). The samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins were transferred to a PVDF membrane (IPVH00010, Millipore). After that, the membranes were incubated with primary antibody overnight at 4°C and secondary antibody for 1 h at room temperature, respectively. Finally, the protein bands were developed by using an enhanced chemiluminescence detection reagent (D045, Bridgen) and imaged by using an Image Lab (BIO-RAD). Primary antibodies against RUNX2 (12556, CST, dilution 1:1000), MMP1 (DF6325, Affinity, dilution 1:1000), E-CADHERIN (20874-1-AP, Proteintech, dilution 1:5000), VIMENTIN (10366-1-AP, Proteintech, dilution 1:5000), and β-ACTIN (AF7018, Affinity, dilution 1:5000) were used. Three independent experiments were conducted.

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).