FIGURE 1.

CN regulates β4 phosphorylation and HD stability. A, effects of CN inhibitor CsA on β4 phosphorylation (Ser¹³⁵⁶). HaCat cells were treated for different lengths of time with CsA (1 μm), EGF (50 ng/ml), or a combination of both and were analyzed by Western blot. B, effects of CsA on HD stability. Quantitative IF analysis of detergent-resistant β4 was used to estimate HD-LS. Cells were treated with CsA (1 μm), EGF at low concentration (1 ng/ml), or a combination of both for the indicated time intervals and then detergent-extracted, fixed, and processed for IF using β4 Ab to measure fluorescence-integrated density per cell. C, representative IF images quantified in B. Bar = 10 μm. D, effects of CsA on EGF-induced migration. Cell migration was tested using wound healing assays in the presence or absence of EGF (1 ng/ml) and/or CsA (*, p < 0.05 versus control; **, p < 0.05 versus EGF or CsA). E, effects of CN-siRNA on β4 phosphorylation. HaCat keratinocytes were transfected with CN- or negative control siRNA and analyzed after 48 h for β4 phosphorylation by Western blot. F, effects of CN-siRNA on HD-LS. Control or CN-siRNA transfectants were analyzed for HD-LS abundance using quantitative IF (*, p < 0.05 versus control siRNA; **, p < 0.05 versus control siRNA+EGF or CN-siRNA). G, effects of CN-siRNA on keratinocyte migration using wound healing assays. HaCat keratinocytes transfected with control or CN-siRNA were grown to confluency for 48 h before performing wound healing assays in the presence of EGF (1 ng/ml) for 18 h. (*, p < 0.05 versus control siRNA; **, p < 0.05 versus control siRNA+EGF or CN-siRNA)