Horseradish peroxidase hrp conjugated anti rabbit igg

In short, cells were washed with cold PBS three times and lysed with radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors for 30 min on ice. The total protein concentration was quantified by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Samples were separated by SDS/polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes (Millipore). Then the membranes were blocked with 5% fat-free milk dissolved in TBST (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, and 0.05% Tween 20) at room temperature for 1 h and incubated with primary antibodies against GAPDH (1:1,000, CW0100M; CWBIO), NAT10 (1:1,000, 13365-1-AP; Proteintech), RUNX2 (1:1,000 dilution, sc-390351; Santa Cruz), and Osterix (1:1,000, ab209484; Abcam) overnight at 4°C. After that, the membranes were incubated with the appropriate secondary antibody, either horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (1:3,000; Beyotime) or anti-mouse IgG (1:3,000; Beyotime), at room temperature for 1 h and visualized with chemiluminescent HRP substrate (Millipore, Billerica, MA, USA). ImageJ software was used to analyze the band intensities.