Cells were lysed directly in 1X SDS sample buffer [1.6% sodium dodecyl sulfate, 0.06M Tris, 5.5% glycerol, and 0.002% bromophenol blue], scraped into tubes, heated at 95°C for 5 minutes, then sonicated until solubilized. For alkaline phosphatase treatment of cell lysates, cells were lysed in RIPA buffer (Thermo Fisher) [25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS] containing protease inhibitor cocktail (Sigma), sonicated, and incubated with 0.1 U/μL cell lysate of calf alkaline phosphatase (Sigma) for 1 hour at 37°C. Protein concentration was determined by Bio-Rad DC Protein Assay (Bio-Rad, Hercules, CA) and then 2-mercaptoethanol was added to a final concentration of 1% to the lysates and heated at 70°C for 20 minutes. An equal amount of protein was added to precast 4–20% gradient or 10% polyacrylamide gels (Bio-Rad) and electrophoresis was performed for 1 hour at 200 V. Protein was transferred to PVDF membrane (Millipore) and blocked for 1 hour at room temperature in 0.2 M Tris, 0.15 M NaCl, 0.01% thimeresol, 0.2% Tween-20, pH 7.4 (TTTBS) for ECL detection or with fluorescent blocker (Millipore) for Odyssey infrared imaging (LI-COR, Lincoln, NE). Membranes were incubated with the following primary antibodies as indicated: mouse anti-Cx43NT1, which recognizes the N-terminal region of Cx43 (Fred Hutchinson Cancer Center, Seattle, WA); rabbit anti-Cx43, which recognizes the C-terminal region of Cx43 (Invitrogen); mouse anti-ZO-1 (Invitrogen); and mouse anti-α-tubulin (Sigma), all diluted in 1% BSA in TTTBS or fluorescent blocker. Secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for enhanced chemiluminescence (ECL) detection and IRDye800 and IRDye680 labeled secondary antibodies (LI-COR) for infrared imaging. Where indicated, blots were stripped for 15 minutes at room temperature (Restore Western Blot Stripping Buffer, Thermo Fisher Scientific, Rockford, IL). Chemiluminescent signal was visualized with ECL substrate (Millipore) followed by detection and capture of 16-bit images with a Bio-Rad FX imager (Bio-Rad), and fluorescent signal was detected with Odyssey infrared imaging system (LI-COR). Densitometry was performed using digital analysis software for ECL (Bio-Rad Quantity One) and fluorescent detection (LI-COR) followed by appropriate statistical analysis for comparisons with GraphPad (GraphPad Software Inc., La Jolla, CA). All forms of Cx43 (phosphorylated and nonphosphorylated) were quantified in densitometry.