4.7. Western Blot Analysis

Protein was harvested from cells in 100 µL lysis buffer (1% (v/v) SDS, 30 mM Na₃VO₄, 1 µM DTT, protease inhibitor cocktail (Sigma-Aldrich) and phenylmethanosulfonylfluoride (PMSF)) prepared in 1xPBS as previously described [52].

Equivalent amounts of protein (30–80 μg) were mixed with 5 µL loading buffer (NuPAGE^® LDS Sample Buffer (4X)) and the volume adjusted to 20 µL with lysis buffer. Samples were mixed for 15 min at 20 rpm followed by brief centrifugation and separated by 4–12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (NuPAGE^® Novex^® Bis-Tris Mini Gels; Thermo Fisher) followed by electrophoretic transfer to a nitrocellulose membrane using an I-Blot transfer system (Invitrogen) following manufacturer’s instructions. Transfer efficiency was determined by staining the blots with Ponceau S (0.1% (w/v) in 5% acetic acid) (Sigma-Aldrich) for 15 s, prior to rinsing in distilled water and probing for relevant protein expression using appropriate primary antibodies as previously described [52]. Membranes were probed with primary antibodies to detect Cx43 (Rivedal polylclonal antibody 1:2000 dilution, kindly gifted by Edward Leithe [53]), GAPDH (mouse monoclonal antibody, Santa Cruz (LOCATION) (1:5000 dilution)) and pSmad3 (rabbit polyclonal antibody Abcam (Cambridge, UK) (1:2000 dilution)) expression as appropriate. Secondary antibodies were IRDye^® 800CW goat anti-rabbit IgG or IRDye^® 680CW goat anti-mouse IgG (Licor 1:15,000 dilution) as appropriate. Blots were developed by exposing the image for a period of 15 s to 5 min according to the intensity of the signal using an Odyssey FC Dual Mode Licor imaging system (LI-COR Biosceinces UK Ltd, Lincoln, UK). Densitometric values were quantified using the Odyssey software. To enable normalisation of the blots and comparison of the effect of different treatments on protein expression, the intensity of the protein bands were compared to the house keeping protein.