Rabbit anti apelin

Cells were treated with ATRA for various times or at different doses and then harvested with 150 mmol/l NaCl, 50 mmol/l Tris/HCl (pH 7.5), 1% NP-40, 0.5% sodium deoxycholic acid, and complete protease inhibitor mixture tablets (Roche Applied Science, Basel, Switzerland). Crude proteins were isolated from HUVECs, separated by SDS/PAGE, and transferred on to PVDF membranes (Millipore, Billerica, MA, U.S.A.). Membranes were blocked with 5% milk in TBS with Tween 20 (TTBS) for 2 h at 37°C and then incubated overnight at 4°C with the following primary antibodies: 1:500 rabbit anti-apelin (GeneTex, U.S.A.), 1:800 rabbit anti-RARα, 1:800 rabbit anti-RARβ, and 1:800 rabbit anti-RARγ (Abcam), and 1:1000 mouse anti-β-actin (Santa Cruz Biotechnology, CA, U.S.A.). After incubation with the appropriate secondary antibody, the immunoreactive signals of antibody antigens were visualized using the Chemiluminescence Plus Western Blot analysis kit (Santa Cruz Biotechnology).

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).