Anti β actin

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.

C2C12 cell lysates were prepared using RIPA buffer containing protease inhibitors, phosphatase inhibitors and dithiothreitol. The protein concentration was measured using the BCA Kit (Beyotime). Western blot analyses were performed with the following primary antibodies: anti-AKT (cat. #4691), anti-p-AKT 473 (cat. #4060), anti-p-AKT 308 (cat. #2965), anti-ERK1/2 (cat. #9102), anti-p-ERK1/2 (cat. #9101), anti-CCND2 (cat. #3741) and anti-JAK1 (cat. #3344) from Cell Signalling Technology (USA) (all at 1:1000), anti-RHOC (1:500, cat. #D260057) from Sangon Biotech (China), and anti-β-actin (1:5000, cat. #A5441) from Sigma-Aldrich (USA). After an overnight incubation at 4 °C, the membranes were incubated for 1 h at RT with the following secondary antibodies: anti-rabbit or anti-mouse IgG-horseradish peroxidase (HRP) (Proteintech, USA). Finally, the protein bands were detected using the Super ECL Detection Reagent (Tanon, China).

The transfected cells were lysed using RIPA Lysis Buffer containing protease inhibitor PMSF on ice. We measured the protein concentration by the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). About 20 μg protein was separated by (SDS‐PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA). The membranes were blocked in 5% non‐fat dry milk with Tris‐buffered saline (TBST) for 1 hour at room temperature, and then incubated the membranes with primary antibody overnight at 4°C, after that they were incubated with HRP‐tagged secondary antibody (1:3000; Sangon Biotech, Shanghai, China) for 1 hour at room temperature. Then, we detected the immunoreactivity by enhanced chemiluminescence system (ECL, Cell Signaling Technologies, Beverly, MA, USA). Image J software was used for the densitometry analysis of each group, β‐actin was used as a loading control. We used the following antibodies: anti‐EGFL6 primary antibody (1:1000, ab140079, Abcam), anti‐β‐actin (1:1000, D110001‐0100, Sangon Biotech), anti‐p‐AKT (1:1000, Ser473, CST), anti‐AKT (1:1000, C67E7, CST). All the experiments were repeated three times.

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.

Cell lysates were prepared by incubating for 30 min in a buffer containing 25 mM Tris (pH 7.5), 75 mM NaCl, 5% glycerol, 2% SDS and protease/phosphatase inhibitors (Sigma) followed by centrifuging at 10,000 g for 10 min at room temperature. The protein concentration was measured using the BCA protein assay kit (Thermo Scientific). Proteins were separated by SDS-PAGE and transferred electrophoretically onto polyvinylidene fluoride (PVDF) membranes (Millipore), which were incubated overnight with the primary antibodies including mouse monoclonal anti-Akt3 (Santa Cruz Biotechnology), mouse monoclonal anti-Flag (Sigma), rabbit polyclonal anti-RIZ1 (Abcam), and rabbit polyclonal anti-β-actin (Sangon Biotech (Shanghai) Co., Ltd.). The bands were quantified by densitometry using ImageJ software.