Scrape-loading dye-transfer assays

MDCK cells were seeded on poly-l-lysine–coated glass coverslips placed into 3.5-cm dishes and grown to 50-75% confluence. In parallel, 3.5-cm dishes not containing coverslips were also seeded with cells. Dishes with and without coverslips were transfected with WT or mutant Cx43 cDNAs, leaving one set of dishes as untransfected controls. At 20 h posttransfection, cells (then close to confluency) in the dishes without coverslips were rinsed in 1× PBS, resuspended in 1× SDS PAGE sample buffer, boiled for 5 min, and used for Western blot analyses to detect expression levels of Cx43. Cells in the dishes containing coverslips were rinsed in 1×x PBS at RT, and 1 ml of 0.05 weight % LY (cat. no. L682; Invitrogen) in 1× PBS was added to each dish. Monolayers were scraped with a sharp razor blade and incubated at 37°C under 5% CO₂ for 10 min. Cells then were rinsed quickly in 1× PBS and fixed in 3.7% formaldehyde for 10 min at RT. Cover slips were rinsed three times in 1× PBS and dye transfer was imaged with a 20× objective on a Nikon Eclipse TE 2000E inverted fluorescence microscope. Scrape-loading dye-transfer assays were also performed with PAECs as a positive control, as described previously (Baker et al., 2008 ). A comparable sample of whole-cell lysate derived from PAECs was analyzed in controls to compare Cx43 expression levels in MDCK cells with the expression level in endogenously Cx43-expressing cells.