Western Blot Analysis of PI3K/AKT Pathway

Whole head and neck tissues and tumors tissues were lysed using a homogenizer (Pro Scientific) in lysis buffer (Roche) containing a pellet of protease inhibitor cocktail. Protein concentration was measured using Pierce 660 nm protein assay reagent (Thermo), and 40 μg of protein lysates were analyzed using the standard Western protocol we have described previously.^{37 (link)} Blots were incubated with primary antibodies at 4°C overnight and secondary antibodies for 1 hour at room temperature. Western blots were imaged by ECL (Biorad) and normalized with respect to GAPDH (Cell Signaling, #5174) expression. The primary antibodies were as follows and purchased from Cell Signaling: p110α (#4255), PDK1 (#3438), pPDK1^Ser241 (#3061), pAKT^Ser473 (#4058), pAKT^Thr308 (#9275), AKT1 (#2938), AKT2 (#4057), pSmad2 ^ser465/467 (#3101), and pSmad3^ser423/425 (#9520). TGFβ1 ELISA was performed as described previously using the R&D systems kit.^{18 (link)}