Fig. 3. Tyrosine 286 is involved in Cx43 stability. (A) Pulse-chase analysis was performed on stably transfected SKHep1 cells expressing either Cx43-WT or Cx43-Y286A, by pulsing for 60 minutes with [³⁵S]-methionine and then chasing for 0, 2, 4 or 6 hour periods. Cells were then lysed, Cx43 immunoprecipitated overnight, and SDS-PAGE and fluorography performed. (B) Pulse-chase experiments were performed as in A, and the level of immunoprecipitated [³⁵S]-labelled Cx43 was quantified from fluorographs using a molecular imager FX. A plot of the mean±s.e.m. percentage of pulse-labelled Cx43 remaining after 0, 2, 4 and 6 hours of chase from four independent experiments fitted using a mono-exponential decay function is shown. ANOVA statistical analysis indicates that the difference in the degradation curves between Cx43-WT and Cx43-Y286A is highly significant (P<0.0001).