Anti β actin and rabbit anti igg

Crude proteins were extracted from liver tissues of mice or LX-2 cells as described previously [23 (link)], resolved by SDS/PAGE, and then transferred to a PVDF membrane (Millipore). Membranes were blocked with 5% (w/v) nonfat dried skimmed milk powder in TTBS buffer (100 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 0.5% Tween 20) for 2 h at 37°C and then incubated overnight at 4°C with the following primary antibodies: 1 : 300 dilution rabbit anti-apelin (GeneTex), 1 : 500 dilution rabbit anti-KLF4 (Abcam), 1 : 2500 dilution rabbit anti-cyclin D1 (Abcam), and anti-β-actin and rabbit anti-IgG (Santa Cruz Biotechnology). After incubation with the appropriate secondary antibody, the immunoreactive signals of antibody-antigens were visualized using a Chemiluminescence Plus Western Blot Analysis kit (Santa Cruz Biotechnology).

Crude proteins were extracted from VSMCs as described previously [32 (link)], resolved by SDS/PAGE, and transferred onto a PVDF membrane (Millipore). Membranes were blocked with 5% (w/v), nonfat, dried skimmed milk powder in TTBS (100 Mm Tris/HCl, pH 7.5, 150 mM NaCl, and 0.5% Tween 20) for 2 h at 37°C and then incubated overnight at 4°C with the following primary antibodies: 1:300 dilution rabbit anti-apelin (GeneTex), 1:500 dilution rabbit anti-KLF4 (Abcam), and anti-β-actin and rabbit anti-IgG (Santa Cruz Biotechnology) antibodies. After incubation with the appropriate secondary antibody, the immunoreactive signals of antibody–antigens were visualized using the Chemiluminescence Plus Western Blotanalysis kit (Tanon Biotechnology).