Fluorchem hd2 imaging system

Protein extraction was performed with 100–150 μL RIPA lysis buffer. Subsequently, 50 μg of each sample was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Primary and secondary antibodies utilized are presented in Supplementary Table S1. The antigen-antibody complex was detected with the ECL Prime Western Blotting System (Cat#GERPN2232, GE Healthcare, Chicago, IL, USA). Protein bands were visualized by the FluorChem HD2 Imaging System (Cell Biosciences, Santa Clara, CA, USA). Protein band intensity was quantified with Image J and normalized to GAPDH. Membranes were stripped and re-probed, as indicated in the supplementary figures.

Membranes were developed with alkaline phosphatase-conjugated secondary antibodies, and signals were visualized using either the Alpha Innotech FluorChem HD2 Imaging System (San Leandro, CA, USA) or the FluoroChem M system (Cell Biosciences, Preston, Australia). The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): Cx26 (sc-130729), Il1β (sc-52012), P-AKT (sc-7985R), AKT (sc-5298), GSK3β (sc-81462), and β-actin (sc-47778). GAPDH (#14C10) was from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies were used at a 1:1000 dilution with Santa Cruz HRP-conjugated anti- mouse and anti-rabbit secondary antibodies at 1:3000. We tested both the change in protein expression by western blot and the change in the extent of coupling by the dye preloading assay which is a functional assay for dye transfer between cells. We utilized parental HeLa cells that do not express gap junctions and HeLa cells that are stably transfected with Cx26 [31 (link)]. We have characterized HeLa26 cells according to growth and migration properties [32 (link), 50 (link)].