Figure 9. p38-δ is required for the TPA-dependent hINV promoter activation.

(a) Knockdown of p38 isoform expression. Keratinocytes were electroporated with 3 μg of control siRNA or siRNA encoding the indicated p38 isoforms per 1 × 106 cells. Total cell extract was prepared at 48 hours after electroporation and 25 μg protein was electrophoresed for immunodetection of each p38 isoform. This experiment was repeated four times and the siRNA-dependent reduction in level ranged as follows: p38-α, 80–90%; p38-β, 70–90%; and p38-δ, 80–90%, as measured by densitometry. (b) p38-δ is required for TPA-dependent hINV promoter activation. At 48 hours after siRNA electroporation, cells were electroporated with 3 μg of endotoxin-free pINV-2473. After an additional 6 hours, the cells were treated with 50 ng ml⁻¹ TPA and luciferase activity was assessed 18 hours later. The results are expressed as the mean±SD. p38-δ siRNA significantly suppresses (P<0.01) TPA-stimulated promoter activity below the activity observed with control siRNA, n = 3. (c) p38-δ knockdown does not influence the level of other p38 isoforms. Keratinocytes were electroporated with 3 μg of control or p38-δ isoform-specific siRNA per 1 × 106 cells. Total cell extract was prepared at 48 hours after electroporation and 25 μg of protein was electrophoresed for immunodetection of each p38 isoform. This experiment was repeated twice with similar results. hINV, human involucrin; siRNA, small interfering RNA; TPA, 12-O-tetradecanoylphorbol-13-acetate.