Western Blot Analysis of Cellular Signaling

Western blot analyses were performed as previously described [18 (link)]. Briefly, cells were lysed and sonicated in RIPA buffer (Thermo Scientific, IL, USA). Equal amounts of protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoretic separation, protein bands were transferred to a Millipore Immobilon-P membrane followed by immunoblotting with antibodies against molecules of interest. The following primary antibodies were used for immunoblotting: anti-Smad2 (Cell Signaling, MA, USA, 1:1000), anti-p-Smad2 (Cell Signaling, 1:1000), anti-Smad3 (Cell Signaling, 1:1000), anti-p-Smad3 (Cell Signaling, 1:1000), anti-Akt (Cell Signaling, 1:1000), anti-p-Akt (Thr308 and Ser473) (Cell Signaling, 1:1000), anti-S6K (Cell Signaling, 1:1000), anti-p-S6K (Cell Signaling, 1:1000), anti-α-SMA (SIGMA, 1:2000), anti-fibronectin (Abcam, 1:1000), anti-vinculin (SIGMA, 1:1000), anti-paxillin (BD Bioscience, 1:1000), anti-MMP1 (Santa Cruz Biotechnology, CA, USA, 1:1000) and anti-GAPDH (Santa Cruz Biotechnology, 1:5000) antibodies. Chemiluminescence detection was performed using ECL reagent (Bio-Rad Laboratories. Inc., CA, USA). The signal intensities of bands were quantified with ImageJ software (NIH).