Hyperfilm ecl kits

Total protein from GC tissues or cultured cells was extracted by ice‐cold RIPA buffer (Thermo Fisher Scientific, Inc.) supplied with 1× protease inhibitor cocktail (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Protein was quantified using the Bradford method. An equal amount (30 μg) of total protein from each sample was separated onto a 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Proteins were then transferred onto a polyvinylidene fluoride membrane (Bio‐rad) followed by blocking by 5% BSA for 1 h at room temperature. Membranes were incubated with primary antibodies for LDHA (1:1000) and β‐actin (1:3000) separately at 4°C overnight. After washing by PBST three times, membranes were incubated with peroxidase‐coupled secondary antibody for 1 h at room temperature. Protein bands were detected using Hyperfilm‐ECL kits (GE Healthcare Biosciences). β‐actin expression was a loading control. Three independent experiments were performed for each analysis.

FLSs were collected and lysed by RIPA lysis buffer (Beyotime Biotechnology, Nantong, China) plus 1 × protease inhibitor cocktail (Sigma‐Aldrich, Shanghai, China). Lysates were centrifuged at 12,000 g at 4°C for 20 min. Equal amount of protein from each group (40 μg) was loaded onto a 10% SDS‐PAGE gel. Proteins were transferred to a nitrocellulose membrane followed by blockading with 5% BSA in TBST buffer. Primary antibodies (1:1000) were incubated with membranes for overnight at 4°C. After complete washing by TBST, membranes were incubated with secondary antibody at room temperature for 2 h. Protein bands were visualized using Hyperfilm‐ECL kits (GE Healthcare Biosciences). Experiments were repeated three times.