Western blot analysis of cultured fibroblasts

Western blot analysis of cultured fibroblasts

Serum-starved confluent HLF cells were coincubated in medium

containing TGFβ1 (10 ng/ml) with or without the indicated concentrations

of PI3K inhibitors for 24 hours. Cells were harvested and

lysed for Western blot analysis. After sodium dodecyl sulfate (SDS)–polyacrylamide

gel electrophoresis and blocking, membranes were

incubated with antibodies to detect pSMAD2Ser465/467 (Cell Signaling

Technology, #3101) or pAktSer473 (Cell Signaling Technology, #4060) or pS6

Ser235/236 (Cell Signaling, #4858S) and signals were visualized with ECL Western Blot Detection Reagents

(GE Healthcare). After stripping, membranes were blocked and

reprobed with antibodies specific for total SMAD2 (Cell Signaling

Technology, #3103) or total Akt (Cell Signaling Technology, #4060).