Figure 5. UVB induced tyrosine phosphorylation of PKD.

Near-confluent primary mouse keratinocytes were irradiated with 30 mJ/cm² UVB, and the control cells were sham-irradiated. The cells were lysed 2 hrs post-UVB exposure and immunoprecipitated using total PKD antibody followed by western analysis with a phosphotyrosine antibody. Aliquots representing one-fifth of the protein quantity immunoprecipitated were blotted with total PKD antibody to serve as the loading control (input). Panel (A) shows a representative blot of 4 separate experiments. (B) Values represent the means ± SEM of 4 separate experiments and are expressed as the –fold over the control vlaue;*p<0.05 versus the control value of 1.0 by a two-tailed Student’s t-test. Note that similar results were observed in experiments conducted in the reverse direction, i.e. immunoblotting for total PKD following immunoprecipitation with anti-phosphotyrosine antibody. Therefore, the results were combined for quantitation in Panel B. (C) Near-confluent primary mouse keratinocytes were irradiated with 30 mJ/cm² UVB (or treated with TPA for 15 minutes as a positive control) and the control cells were sham-irradiated (Con). The cells were lysed 2 hrs post-UVB exposure and western analysis was performed using anti-phosphotyrosine⁴⁶³ and total PKD antibodies. The experiment was repeated with similar results. Shown under the blot are the densitometric values (normalized to the loading control) relative to the average (normalized) control value.