Ab126753

Radio‐Immunoprecipitation assay (RIPA) (containing phenylmethylsulfonyl fluoride) lysis was used for extraction of total protein from tissues or cells. After 30 min of incubation on ice and 10 min of centrifugation (8000g, 4°C), the supernatant was collected for protein concentration detection with a bicinchoninic acid kit. Next, 50 μg protein was dissolved in 2× sodium dodecyl sulfate (SDS) loading buffer and boiled at 100°C for 5 min, followed by SDS‐polyacrylamide gel electrophoresis. Then the protein was transferred onto a polyvinylidene fluoride membrane, after which the membrane was blocked in 5% skim milk for an hour at room temperature. Afterward, the membrane was incubated at 4°C overnight with primary antibodies from Abcam against STIP1 (ab126753, 1:10,000), HSP90 (ab59459, 1:500), Cx43 (ab11370, 1:2000), and β‐actin (ab8226, 1:2000). After washing, horseradish peroxidase (HRP) labeled goat anti‐rabbit immunoglobulin G (IgG) (1:5000, Beijing ComWin Biotechnology Co., Ltd.) was appended to the membrane for 2 h of incubation, followed by treatment with electrochemical luminescence solution for color development. Finally, the protein bands were scanned and analyzed on a gel imager, and gray quantification of the western blot images was analyzed by the software Image Pro Plus 6.0 (Media Cybernetics). Each experiment was repeated three times.

Cells were lysed with precooled RIPA lysis and 15‐min centrifugation (at 14,000g and 4°C) was performed. Then the supernatant was removed to a new tube. Anti‐STIP1 (ab126753, 1:20, Abcam), anti‐Cx43 (ab235585, 1:30, Abcam), anti‐HSP70 (ab2787, 1:50, Abcam), or anti‐HSP90 (ab59459, 1:50, Abcam) was added to 1 mL cell lysate, and IgG antibody (ab172730, 1:100, Abcam) was added to the NC group. The antigen‐antibody mixture was slowly shaken on a shaker overnight at 4°C or for 2 h at room temperature. After that, the mixture was added with 100 μL Protein A/G agarose beads (prepared in PBS with a concentration of 50%) and incubated at 4°C overnight or for 1 h at room temperature. The agarose bead‐antigen‐antibody complex was obtained after centrifugation at 14,000 rpm for 5 s. Next, the complex was rinsed three times with precooled RIPA (800 μL) and suspended with 2× loading buffer (60 μL). The sample was boiled for 5 min and centrifuged to collect the remaining agarose beads, and the supernatant was boiled again for 5 min and then electrophoresed. Protein expression was measured by western blotting.