Fig. 4. Differential responses of wild-type Cx43 and Cx43-Y286A to both proteasomal and lysosomal inhibitors. (A) The stable cell lines, Cx43-WT and Cx43-Y286A, were treated for 3 hours with the proteasome inhibitor lactacystin (10 µM) or the lysosomal inhibitors leupeptin (10 µM) or NH₄Cl (10 mM). Western blot analysis was then performed on the cellular lysates using either an anti-Cx43 (top panel) or a ß-actin (bottom panel) antibody. Steady-state levels of Cx43-Y286A were consistently greater than Cx43-WT, therefore reduced fluorography was performed for comparative analysis. (B) Quantitation of Cx43 protein levels in response to pharmacological agents. Experiments were performed as in A, and the amounts of Cx43-NP (left panel) and Cx43-P1,P2 (right panel) detected by western blot fluorography were quantified using a molecular imager FX. Mean ± s.e.m. values of normalised Cx43-WT (grey columns) and Cx43-Y286A (black columns) expression levels are shown (n=3 independent experiments). Asterisks represent P<0.01 versus control determined using the Student's t-test.