“…HBF cultures were lysed as described previously [ 23 , 30 ], and the protein content in the supernatant was determined using the Bradford method. Protein samples (30 µg/lane) were electrophoresed on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Bio Rad, Hercules, CA, USA) as described previously [ 23 , 57 ]. Next, after blocking, the membranes were incubated overnight at 4 °C with primary antibodies: rabbit polyclonal IgG against p(Ser467)-Smad2 (1:500), rabbit polyclonal IgG against Cx43 (1:2000), mouse monoclonal IgG against α-SMA (1:2000), mouse monoclonal IgG against vinculin (1:1000), mouse monoclonal IgG against β-tubulin (1:1000), and mouse monoclonal IgG against glyceraldehyde-3-phosphate dehydrogenase GAPDH (1:3000), all from Sigma-Aldrich, St. Louis, MO, USA; rabbit monoclonal IgG against Smad2 (D43B4), rabbit monoclonal IgG against p(Ser465/467)-Smad2 (138D4), rabbit monoclonal IgG against Smad3 (C67H9), rabbit monoclonal IgG against p(Ser423/425)-Smad3 (C25A9), rabbit polyclonal IgG against Focal adhesion kinase (FAK), rabbit polyclonal IgG against p(Thr202/Tyr204)-Erk1/2, and rabbit polyclonal IgG against Erk1/2 (all: 1:500; Cell Signaling Technology ® Danvers, MA, USA).…”