Fig. 1. Mutation of tyrosine 286 to alanine affects Cx43 steady-state levels. (A) The amino acid sequence of Cx43 spanning residues 280-291. The putative PY-motif and tyrosine-based sorting signal are indicated and their consensus sequences displayed. (B) Transient expression of human Cx43 constructs in SKHep1. Cells were transfected with cDNA plasmids encoding either Cx43-WT, Cx43 with a substitution of tyrosine to alanine at position 286 (Cx43-Y286A) or neither (untransfected). Following expression (48 hours), western blot analysis was performed on cellular lysates using either an anti-Cx43 antibody (top panel) or a ß-actin antibody (bottom panel) to control protein loading. Cx43 was detected in its nonphosphorylated (NP) and phosphorylated (P1, P2) forms (indicated by arrows) and was determined by treating cellular lysates with CIP before western blot analysis.