Hrp conjugated anti rabbit secondary antibody

After drug treatment, cells were solubilized in lysis buffer containing 0.125 M Tris-HCl, PH6.8, 4% SDS, 20% glycerol, and 2% 2-mercaptoethanol and analyzed immediately or stored as being frozen at −20°C. Proteins were separated on 8% SDS-polyacrylamide gels and transferred to a PVDF membrane (Millipore, Billerica, MA). The PVDF membrane was blocked with 5% fat-free milk in Tris-buffer saline/0.1% Tween 20 (TBS-T) and then incubated in the primary antibodies diluted in 2.5% fat-free milk in TBS-T over night at 4°C. The primary antibodies were as follows: anti-PGC-1α (Sangon Biotech, D162041, 1 : 1,000), anti-β-actin (Sangon Biotech, D110001, 1 : 5,000), anti-phospho-CREB (Cell Signaling, Danvers, MA, 9198, 1 : 1,000), and anti-CREB (Cell Signaling, 9197, 1 : 5,000). After that, the PVDF membrane was rinsed with TBS-T and incubated for 2 h at room temperature in peroxidase (HRP)conjugated anti-rabbit secondary antibody (Sangon Biotech, D110058, 1 : 5,000), diluted in 2.5% fat-free milk in TBS-T. After extensive washing with TBS-T, the immune complexes were visualized using the enhanced chemiluminescence (ECL) kit (Vazyme). The intensities of bands in control and samples, run on the same gel and under strictly standardized ECL conditions, were compared on an image analyzer, using a calibration plot constructed from a parallel gel with serial dilutions of one of the sample.

The cultured cells were collected, and protein content was determined by Bradford method. Proteins (~20 μg) were separated on 8% SDS‐polyacrylamide gels and transferred to a PVDF membrane. The PVDF membrane was blocked with 5% fat‐free milk in tris‐buffer saline/0.1% tween 20 (TBS‐T) and then incubated in the primary antibodies diluted in 2.5% fat‐free milk in TBS‐T over night at 4°C. The primary antibodies were anti‐phospho‐CREB (1:1000, Cell Signaling Technology, 9198, Cat# ABIN461313), anti‐CREB (1:1000, Cell Signaling Technology, 9197, Cat# 27‐321), anti‐PGC‐1α (1:1000, Sangon Biotech, D162041, Cat# sc‐518025), anti‐β‐actin (1:5000, Sangon Biotech, D110001, Cat# 130‐120‐277), and anti‐GAPDH (1:5000, Sangon Biotech, D110016, Cat# JM‐3777‐100). After that, the PVDF membrane was rinsed with TBS‐T and incubated for 2 h at room temperature in peroxidase (HRP)‐conjugated anti‐rabbit secondary antibody (1:5000, Sangon Biotech, D110058), diluted in 2.5% fat‐free milk in TBS‐T. After intensive washing with TBS‐T, the immune complexes were visualized using the enhanced chemiluminescence (ECL) method (Vazyme). The intensities of bands in control and samples, run on the same gel and under strictly standardized ECL conditions, were compared on an image analyzer, using a calibration plot constructed from a parallel gel with serial dilutions of one of the sample.