Figure 2. mRNA and protein expression of upstream kinases in the buccal pouch tissues during the stepwise progression of HBP carcinomas (mean ± SD; n = 3).

(A) mRNA expression level of PI3K and ERK in control and DMBA painted animals as determined by kinetic PCR. The fold change in transcript expression for each gene was determined using the 2−ΔΔCt method. Data are the mean ± SD of three independent experiments. Statistical significance was determined by the Mann–Whitney test (p < 0.05). (B) Western blots showing overexpression of PI3K, p-Akt^Ser473 and p-ERK1/2^{Thr202/Tyr204} in DMBA painted animals from 0 week to 16 weeks. Gapdh was used as loading control. Phosphorylated proteins are normalized by their unphosphorylated forms. (C) Background subtracted protein bands quantified and normalized to Gapdh. Phosphorylated proteins are normalized by their unphosphorylated forms. Each bar represents the protein expression ± SD of three determinations. (D) Total p-Akt^Thr308 as determined by ELISA showing a sequential increase from 0 to 16 weeks. (E) Representative photomicrographs of immunohistochemical staining of pAkt^Ser473 in control and experimental animals (×20). ^♣p < 0.05 versus control.