Western blot was performed as previously described [12 (link), 13 (link)]. Briefly, lysates were prepared in RIPA buffer with protease inhibitor cocktail (Sigma). The concentrations of total proteins were measured by Bradford protein assay (Bio-Rad). Total lysates were loaded on SDS-PAGE, and electrotransferred onto PVDF membrane. The primary antibodies rabbit anti-p-SMAD2 (Cell signaling), rabbit anti-p-SMAD3 (Cell signaling), and mouse anti-GAPDH (Millipore) were used.
After washing with PBST, the membranes were incubated with horseradish peroxidase coupled secondary antibodies. The signals were visualized using ECL reagents (Amersham).
After washing with PBST, the membranes were incubated with horseradish peroxidase coupled secondary antibodies. The signals were visualized using ECL reagents (Amersham).
