Horseradish peroxidase conjugated anti mouse or anti rabbit igg secondary antibodies

Total proteins were extracted with RIPA lysis buffer containing protease inhibitors. The protein concentration was determined using BCA reagent kit (Invitrogen, Carlsbad, USA). Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, USA). Membranes were blocked with 5% skim milk, and then incubated with primary antibody at 4°C overnight. Membranes were then washed with Tris-buffered saline containing Tween 20, incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Santa Cruz, Santa Cruz, USA) for 1 h at room temperature, and developed with enhanced chemiluminescence kit (Microwell, Billerica, USA). β-Actin served as the loading control. Antibody against pVHL was from BD Biosciences (San Jose, USA). Anti-PHD3 antibody was from Novus Biologicals (Littleton, USA). The pan-hydroxylated proline antibody was obtained from Advanced Targeting Systems (San Diego, USA). Antibodies against PAX2, HA and Myc were products of Santa Cruz (Santa Cruz, USA). Anti-β-actin and anti-Flag antibodies were from Sigma (St Louis, USA) and Abmart (Shanghai, China), respectively.

Total cell lysate from 5×10⁶ leukemia cells was prepared by direct lysis of cells with RIPA buffer [20 mM Tris-Cl (pH 7.5) 150 mM NaCl, 1% Triton X-100, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.5 mM PMSF]. The amount of total protein was assayed using a Pierce^® BCA Protein Assay (Pierce; Thermo Fisher Scientific, Inc.). The lysates (20 µg/lane) were electrophoresed on 10% polyacrylamide gel, and subsequently transferred to an Immobilon membrane (EMD Millipore). The membrane was blocked in 5% bovine serum albumin at 4°C (Sigma-Aldrich; Merck KGaA) for 1 h, and then incubated with primary antibodies at 4°C overnight against mouse Hoxa11 (1:5,000; cat. no. NBP1-80228; Novus Biologicals, Ltd.), β-actin or Gapdh (1:10,000; cat. nos. sc-47778 or sc-32233; Santa Cruz Biotechnology, Inc.), followed by incubation for 2 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (1:5,000; cat. nos. C04001 or C04003; Croyez Bioscience Co., Ltd.). Western blots were developed with a Western Lightning Plus ECL kit (PerkinElmer, Inc.) and the images were visualized by Analytik Jena™ UVP ChemStudio PLUS and VisionWorks™ software (version 9.0; Analytik Jena US LLC).

Myocardial tissues and H9C2 cells were homogenized in ice-cold RIPA buffer (25 mmol/L Tris·HCl [pH 7.6], 150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail, and phosphatase inhibitor cocktail). The homogenate sample was sonicated briefly before centrifugation at 13,500 ×g for 30 min at 4℃. The BCA assay kit (Pierce, Rockford, IL, USA) was used to quantitate protein. Protein (50~100 µg) was separated by SDS-PAGE on a 10~12% polyacrylamide gel. Proteins were then transferred to nitrocellulose membranes (GE Healthcare, Chalfont St. Giles, UK) and blocked in 5% skim milk for 2 h at room temperature. Membranes were then probed with mouse or rabbit polyclonal antibodies to Dcn, Smad2, or pSmad2 (Abcam), or with mouse monoclonal antibodies to α-tubulin (Sigma-Aldrich) at a 1:500 dilution overnight at 4℃. Following three washes in PBS with Tween 20 (PBS-T, pH 7.4), membranes were incubated with horseradish peroxidase-conjugated antimouse or anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) at a 1:2000 dilution for 2 h at room temperature. Following washing with PBS-T three times, a western blotting detection kit (Abclon Inc., Seoul, Korea) was utilized to visualize the antibody-bound proteins. These experiments were performed in triplicate.