Fig. 2. The tyrosine-based sorting signal, adjacent to the PY-motif, is the major element affecting Cx43 levels. (A) Transient expression of Cx43 mutant constructs in SKHep1 cells. Mutant Cx43 plasmids were developed by exchanging proline with leucine at position 283 (Cx43-P283L), glycine with alanine at position 285 (Cx43-G285A), and valine with aspartate at 289 (Cx43-V289D). Cells were transfected with these plasmids, as well as Cx43-WT and Cx43-Y286A, cellular lysates prepared (48 hours after transfection) and western blot analysis performed using an anti-Cx43 antibody (top panel) or a ß-actin antibody (bottom panel) to control protein loading. (B) Quantitation of transfected Cx43 transiently expressed in SKHep1. Experiments were performed as in A, and the levels of total Cx43 and ß-actin detected by western blot fluorography quantified on a molecular imager FX. The Cx43:actin ratio value was determined for each construct and displayed as mean±s.e.m. (n=4 separate experiments). Asterisks indicate differences at P<0.01 vs control as determined by the Student's t-test.