Fig. 5. The lysosomal inhibitor, NH₄Cl, affects wild-type, but less efficiently, mutant (Y286A) Cx43 turnover. (A) Pulse-chase analysis was performed on either Cx43-WT cells or Cx43-Y286A cells by pulsing for 60 minutes with [³⁵S]-methionine, and then chasing for 0, 3 or 6 hour periods in the absence (control) or presence of the lysosomal inhibitor, NH₄Cl. (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, 3 and 6 hours of chase (n=4 independent experiments), fitted using a mono-exponential decay function is shown. ANOVA statistical analysis reveals that the difference in the degradation curves between Cx43-WT (control and NH₄Cl) is highly significant (P<0.0001), in contrast to the Cx43-Y286A (control and NH₄Cl), which is significant (P=0.0303).