Ecl kit

Proteins were isolated from cells using RIPA lysis buffer (Thermo Scientific) and quantified with a BCA assay kit (Bio‐Rad, Hercules, CA, USA). Equal amounts of protein samples (20 μg) were dissolved by 10% SDS-PAGE (Bio-Rad) and blotted onto polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Beijing, China). Membranes were blocked with 5% defatted milk and incubated at 4°C overnight with primary antibodies against fibroblast growth factor 2 (FGF2, ab208687), vascular endothelial growth factor (VEGF, ab46154), angiopoietin 1 (Ang1, ab183701), β-actin (ab115777) (all from Abcam, Cambridge, MA, USA) and GMFG (13625-1-AP; Proteintech, Chicago, IL, USA), followed by incubation with the secondary antibody (ab7090, Abcam) at room temperature for 2 h. The blots were visualized with the ECL kit (Vazyme, Nanjing, China) and quantified with ImageJ software (GE Healthcare).

Cells were digested with 0.05% trypsin and 0.02% EDTA for 30 s – 2 min. Then, a complete medium was added to terminate the digestion. The supernatant was centrifuged at 4°C 800 r/min, and added 200 μL 1% SDS protease inhibitor to lyse cells, and the lysate was repeatedly pumped (on ice bath). Protein concentration was quantified following the instructions of Pierce protein assay kit. Before use, freeze-thaw samples on ice, take a certain volume (including 50 μg protein) into a clean Eppendorf tube and add 4 μL 5 × SDS loading buffer, 1% SDS to 20 μL. Denatured at 95°C for 5 min, the samples were placed on ice and loaded as soon as possible. 10% SDS-PAGEs were used to separate the proteins, which were then transferred to the ECL membranes (100 V, 1 h), sealed with 10% no-fat milk for 2 h at 5% PBST, and then incubated with primary antibodies overnight at 4°C. PBST solution was used to wash membranes three times, which were then reacted with horseradish peroxidase labeled Goat anti-mouse Ig at room temperature for 1.5 h. Finally, membranes were exposed using an ECL kit (Cat # E411-03, Vazyme, Nanjing, China) to detect protein expression. GAPDH served as an endogenous control.

Total proteins of cultured cells or tissues were obtained in the lysis buffer, separated by the SDS-PAGE, and then transferred into PVDF membranes. The membranes were blocked by 5% nonfat milk at room temperature for 1 h, and subsequently incubated with different primary antibodies at 4 °C overnight and appropriate secondary antibodies at room temperature for 1 h. The immunoreactive signals were visualized by an ECL kit (Vazyme, E411-04/05).

The total protein of cell samples was extracted using RIPA lysis buffer (Beyotime, P0013C) containing phenylmethanesulfonyl fluoride (1 mm). After quantification by a BCA kit (Beyotime, P0012S), the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime, P0015) was added to the total protein lysate, and the mixed buffer was boiled at 100°C for 5 min. Total proteins (40 μg) of each group were added to the gels and separated through electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes. After being blocked in 5% BSA-PBST for 2 h, the immunoblots were immersed in 5% BSA-PBST, which contained primary antibodies overnight at 4°C. Next day, the bands were washed three times with 0.05% tween-PBS (PBST) and then combined with the corresponding HRP conjugated secondary antibodies and detected by an ECL kit (Vazyme, E411-04) and analyzed by ImageJ software.

The extraction of total protein was performed using RIPA buffer (Solarbio). The BCA Protein Quantification Kit (Vazyme) was employed for protein quantification. Next, 20 μg proteins were split via sodium dodecyl sulfonate-polyacrylamide gel (Solarbio) and blotted onto 10% polyvinylidene difluoride membranes (Sigma-Aldrich). After that, the membranes were mixed in 5% slim milk for 1 h at indoor temperature and then incubated with primary antibodies against GAPDH (ab9485; Abcam, Cambridge, MA, USA) or VASP (bs229624; Abcam) overnight at 4 °C followed by interaction with secondary antibody (ab205719; Abcam) for 1.5 h at indoor temperature. The ECL kit (Vazyme) was employed for chemiluminescence visualizing.

Cells were digested with 0.05% trypsin and 0.02% EDTA for 30 s – 2 min. Then complete medium was added to terminate the digestion. The supernatant was centrifuged at 4°C 800 r/min, and added 200 μL 1% SDS protease inhibitor to lyse cells on ice bath. The protein concentration was quantified following the instructions of Pierce protein assay kit. Before use, freeze-thaw samples on ice, take a certain volume (including 50 μg protein) into a clean Eppendorf tube and add 4 μL 5 × SDS loading buffer, 1% SDS to 20 μL. Denatured at 95°C for 5 min, the samples were placed on ice and loaded as soon as possible. 10% SDS-PAGEs were used to separate the proteins, which were then transferred to the ECL membranes (100 V, 1 h), sealed with 10% no-fat milk for 2 h at 5% PBST, and then incubated with primary antibodies overnight at 4°C. PBST solution was used to wash membranes three times, which were then reacted with horseradish peroxidase labeled Goat anti mouse Ig at room temperature for 1.5 h. Finally, membranes were exposed using an ECL kit (Cat # E411-03, Vazyme, Nanjing, China) to detect protein expression. GAPDH served as an endogenous control. For protein ubiquitination assay, MG132 was added into cells 6 h before collecting the samples. For protein stability assay, cycloheximide (CHX) (Cat # 40325ES03, YEASEN, Shanghai, China) was used to inhibit protein synthesis.

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.