Figure 3. UVB did not increase phosphoserine^744/748 PKD phosphorylation (in particular phosphoserine⁷⁴⁴ PKD transphosphorylation) in primary mouse keratinocytes, but enhanced serine⁷⁴⁸ (serine⁷⁴² in human) autophosphorylation.

(A) Near-confluent primary mouse keratinocytes were irradiated with 30 mJ/cm² and 60 mJ/cm² UVB, and the control cells were sham-irradiated. The cells were lysed at various time points after exposure and processed for western blotting employing a Cell Signaling antibody against phosphoserine^744/748 PKD, which primarily recognizes phosphoserine⁷⁴⁴ as well as an antibody recognizing total PKD. Actin served as the loading control, and TPA (100 nM) stimulation for 30 minutes served as a positive control. Illustrated is a blot representative of 3 separate experiments. (B) Near-confluent primary mouse keratinocytes irradiated with 30 mJ/cm² UVB were lysed 2 h post-UVB and processed for western blotting. Control cells (Con) were sham-irradiated, and a 15-minute treatment with TPA (100 nM) was used as a positive control. Analysis was performed with an Abcam antibody recognizing autophosphorylated phosphoserine⁷⁴² (phosphoserine⁷⁴⁸ in mouse). Heat shock protein 90 (Hsp90) served as the loading control. Shown under the blot are the densitometric values (normalized to the loading control) relative to the average (normalized) control value obtained with the Alpha Innotech gel imaging system. The experiment was repeated with similar results.