Fig. 3

PKCα is the key player in PMA-induced NF-κB activation. Three pairs of small interfering RNA against PKCα were designed, and the knockdown efficiencies were analyzed by real-time PCR (b) and western blot (a). c Cells were treated/transfected with DMSO/negative control (NC), PMA/NC or PMA/siPKCα for 12 h, and protein expression of PKCα and nuclear/cytoplasmic p65 were detected by western blot. The experiment was repeated three times with each pair of siRNAs against PKCα, and similar results were obtained. A dual luciferasy reporter assay was performed in parallel to confirm the result (g). d Protein expression levels were normalized to Tubulin/Histone, and the band intensities were calculated and analyzed. (e) Cells were pretreated with DMSO, Gö6976 (100 nM) or Sotrastaurin (100 nM) for 1 h and then challenged with PMA (10 ng/ml) for 12 h. Cells without any treatment were used as the blank control. Protein expression of PKCα, p-PKCα and nuclear/cytoplasmic p65 were detected by western blot, normalized and analyzed against the internal control (f). Also a dual-luciferasy reporter assay was performed in parallel to confirm the reslut (h). The gels were run under the same experimental conditions. The band intensities were calculated using the ImageJ 1.46r software. β-Tubulin was used as an internal control for the total protein measurements, and Histone was used as a nucleoprotein reference. The ratio of the target gene to β-Tubulin/Histone was used to conduct the statistical analysis. *P < 0.05 and **P < 0.01, as determined by Student’s t-test