Proteinase and phosphatase inhibitors

Whole cell lysates were extracted using RIPA Lysis Buffer with proteinase and phosphatase inhibitors (Santa Cruz Biotechnology). Extraction of cytoplasmic and nuclear proteins was performed using the Cell Fractionation Kit (Cell Signaling, Danvers, MA, USA). Protein concentrations of lysates were measured by the Pierce™ BCA Protein Assay (ThermoFisher Scientific) using a FLUO Star OPTIMA microplate reader (BMG, Cary, NC). Equal amounts of proteins were loaded onto gradient 8–10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Western blotting was performed following standard procedure. The information of antibodies is provided in Additional file 1: Table S2. The ChemiDoc™ XRS+ Imaging System (Bio-Rad) was used to capture western blot signals. Bands’ intensity was measured using Image lab software (Bio-Rad). The quantification of the proteins was normalized to the intensity of β-actin of the same sample.

Total protein lysates were prepared using standard RIPA lysis buffer (Sigma-Aldrich) with proteinase and phosphatase inhibitors (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The total protein content was estimated by the Pierce™ BCA Protein Assay Kit (Thermo Scientific) following the manufacturer’s protocol. Protein lysates (50 μg) were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% skim milk solution in TBST buffer for 1 h at approximately 24 °C and incubated with polyclonal Connexin 43 (1:500; Proteintech Group, Wuhan, China) and polyclonal GAPDH (1:2000; Proteintech Group, Wuhan, China) primary antibodies at 4 °C overnight. The membranes were incubated with horseradish peroxidase (HRP)‐conjugated secondary antibody (1:5000, GE, HyClone) at 37 °C for 2 h The protein bands were visualized with enhanced chemiluminescence (ECL; Advansta) and detected using a ChemiDocTM MP imaging system (Bio‐Rad). The protein bands were then scanned using Image LabTM Software Version 4.1.