Bca protein extraction kit

The peritoneum samples of the piglets were collected, dissociated by RIPA lysis buffer supplemented with protease inhibitor mixture and centrifuged at 12,000 g for 15 min at 4 °C. The total protein was measured with BCA protein extraction kit (Beyotime). Subsequently, samples with the same amount of protein (80 μg) were fractionated using 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocked with 5% skimmed milk for 3 h, the PVDF membranes were incubated with special primary antibody (containing 5% BSA TBS-T solution, 1:1000 dilution, rabbit anti-β-actin, anti-PKC-α, anti-p-PKC-α, Cell Signaling, Danvers, MA, USA; anti-MLCK, anti-MLC-2 and anti-p-MLC-2, Abcam, Shanghai, China) at 4 °C overnight, and then incubated with the corresponding HRP labeled secondary antibodies (1:4000 dilution) at 37 °C for 3 h. Protein level was determined using the enhanced chemiluminescent (ECL) reagent (Beyotime) and the images were captured with a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). Quantitative analysis was carried out using FluorChem FC2 (Alpha Innotech, San Leandro, CA, USA). The β-actin was used as the inner loading control. Gray value was analyzed and the relative expression level of protein was obtained.

A549 and H460 cells were collected and put on ice with lysis buffers (Tris–HCl pH-6.8, glycerin, 10% SDS, DTT, and protease inhibitors), and the cells were ultrasonically broken using a cell crusher. After centrifugation at 12,000 rpm at 4 °C for 10 min, the supernatant was collected and quantified using a Beyotime Biotechnology (BCA) protein extraction kit. The protein was stored in a refrigerator at -80℃ for later use.