Pvdf filter membranes

Total protein from each group was extracted and the protein concentration was measured using the Bradford DC protein assay (Bio-Rad, Hercules, CA, USA). 25 μg protein from each sample was separated using SDS–polyacrylamide gel electrophoresis (10 %) and blotted onto polyvinylidene difluoride (PVDF) filter membranes (Whatman). The membranes were blocked in 5 % skim milk for 1 h, washed four times with Tris-buffered saline containing Tween 20 (TBST) at room temperature and then incubated overnight at 4 °C with diluted primary antibodies (rabbit anti-human Cthrc1/TIMP-1/MMP-2/MMP-9 antibody, 1:500, Santa Cruz). Following extensive washing with TBST, membranes were incubated with secondary antibodies at room temperature for 1 h (goat anti-rabbit IgG, 1:1000, Santa Cruz). After four 15 min washes with TBST at room temperature, immunoreactivity was visualized by enhanced chemiluminescence. GAPDH expression (Santa Cruz Biotechnology) served as an endogenous reference.

Total proteins were extracted from the transfected cells at 48 h after transfection and then were subjected to SDS-PAGE (SDS/polyacrylamide (10 %) gel electrophoresis) and transferred electrophoretically onto polyvinylidene difluoride (PVDF) filter membranes (Whatman). The membranes were blocked in 5 % skim milk for 1 h, washed four times with Tris-buffered saline containing Tween 20 (TBST) at room temperature, then incubated overnight at 4 °C with diluted primary antibody (rabbit anti-human RGMB antibody, 1:500, Santa Cruz Biotechnology). Following extensive washing with TBST, the membranes were incubated with secondary antibody (goat anti-rabbit IgG, 1:2000, Santa Cruz Biotechnology) for 1 h. After four washes (15 min each) with TBST at room temperature, the immunoreactivity was visualized by enhanced chemiluminescence. The antibody against GAPDH (Santa Cruz Biotechnology) served as an endogenous reference.