Horseradish peroxidase conjugated secondary antibody

Total protein was extracted from tissues and cells using RIPA buffer and the quality of protein was evaluated using BCA protein assay kit (Pierce, Rockford, IL, USA). The extracted proteins were resolved on 14% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. Thereafter, the membranes were blocked with 5% fat-free milk, incubated with primary antibodies against TGF-β1 (catalog number: ab92486; Abcam, Cambridge, MA, USA), VCAM-1 (catalog number: ab174279; Abcam), Smad3 (catalog number: ab122028; Abcam), and β-actin (catalog number: BM0627; Boster, Wuhan, China) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (catalog number: BA1050; Boster) for 1 h at 25 °C. Bands were developed using a BeyoECL Moon kit (Beyotime Biotechnology, Shanghai, China) for visualization. The experiment was independently repeated at least three times.

Western blot analysis was conducted as described before (Dong et al., 2016 (link), 2017 (link)). The cells, seeded in 6-well plates and have been treated or not, were lysed with RIPA lysis solution (Boster) containing broad spectrum phosphatase inhibitors and PMSF (Boster). Total concentration of proteins was measured by BCA assays (Boster, Wuhan, China). The electrophoresis of proteins was conducted in 10% SDS-polyacrylamide gel and then the proteins were transferred to the PVDF membranes (Millipore, MA, United States). 5% bovine serum albumin was used for blocking of the membranes, then the membranes were incubated with respective antibodies overnight. After washed, the membranes were incubated at 25°C for 1 h with horseradish peroxidase-conjugated secondary antibodies (Boster). Subsequently, enhanced chemiluminescence (Boster) was used to visualize the proteins, and images were acquired through ChemiDoc^TM XRS+ System with Image Lab^TM Software (Bio-Rad Laboratories, CA, United States).

Immunoblot analysis was done in RAW264.7 cells as described earlier (Guan et al., 2013 (link), 2015 (link)). The primary antibodies included mouse anti-GAPDH and anti-β-Actin were from Boster (Wuhan, China). Cells were lysed using the protein extraction reagent RIPA (Boster, Wuhan, China) supplement with 1 mM PMSF. The protein concentration was determined using the BCA assay. An equivalent amount of protein was resolved by 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Billerica, MA, United States). Subsequently, membranes were blocked and immunoblotted with individual antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Boster, Wuhan, China). The immunoreactive proteins were visualized using enhanced chemiluminescence (Boster, Wuhan, China) and captured by a scanner (ChemiDoc MP, Bio-Rad, United States).

Proteins were extracted from C2C12 cells using lysis buffer (Sigma, Louis, Mo, USA) according to the manufacturer’s instructions. The wells of a 10% SDS-polyacrylamide gel were loaded with equal amounts of protein (20 μg), samples were then electrophoretically separated and finally transferred to a PVDF membrane (Bio-Rad, CA, USA). The membranes were hybridized with a primary antibody against Foxj3 (Santa Cruz, Santa Cruz, CA, USA), mitochondrial cytochrome c oxidase subunit II (COX II), voltage dependent anion channel (VDAC) and β-Actin (Boster, Wuhan, China), and incubated overnight at 4°C. Membranes were washed and treated with horseradish peroxidase-conjugated secondary antibodies (Boster), enzyme activity was then visualized with DAB substrate solution (Boster).

Immunoblot analysis was performed as described previously52 (link)53 (link). Cells were lysed using the protein extraction reagent RIPA (BOSTER, Wuhan, China) supplement with 1 mM PMSF. The protein concentration was determined using the BCA assay. An equivalent amount of protein were resolved by 10% SDS-PAGE gel and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Subsequently, membranes were blocked and immunoblotted with individual antibodies. The membranes were washed and incubated with horseradish peroxidase–conjugated secondary antibodies (BOSTER, Wuhan, China, dilution 1:5000). The immunoreactive proteins were visualized using enhanced chemiluminescence (BOSTER, Wuhan, China). The protein bands were captured using ChemiDoc™ XRS+ System with Image Lab™ Software (BIO-RAD).

Total proteins of bovine fat cells were extracted using radioimmunoprecipitation assay buffer (RIPA) buffer with 1% PMSF (Solarbio, Beijing, China) after transfection for 24 h or induction for 6 days after transfection. The protein concentration was determined by a bicinchoninic acid (BCA) kit (Beyotime, Shanghai, China), a 5× protein loading buffer (containing mercaptoethanol) was added to proteins at a ratio of 1:4, and then the sample was heated in boiling water for 3–5 min. Proteins were then separated by SDS-polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose membranes and blocked with milk powder solution for 2 h at room temperature. The membranes were then incubated overnight with the primary antibody of anti-CDK2, anti-PCNA, anti-PPARγ, anti-C/EBPβ, and anti-β-actin (Wanlei Bio, Shenyang, China). The membranes were then washed with PBS-Tween 20 and incubated for 1.5 h with horseradish peroxidase-conjugated secondary antibodies (Boster, Pleasanton, CA, USA). Finally, the membranes were imaged with an enhanced chemiluminescence (ECL) kit (Solarbio, Beijing, China) and quantified with the ImageJ program (Bio-Rad, USA).

Cultured cells were treated as above and lysed in 20 μL of cell lysis buffer containing 1 mm phenylmethanesulfonyl fluoride (PMSF). Samples from these cell lysates were denatured and subjected to 17% SDS-PAGE. Proteins were transferred to PVDF membranes by 2 h electroblotting. Membranes were blocked in 5% nonfat dry milk for 1 h at RT and then incubated overnight at 4°C with primary antibodies. Following each incubation, the membrane was washed extensively with TBS containing 0.05% Tween-20 (TBST buffer) three times, probed overnight with anti-SOCS3 (Abcam, CA, USA) and anti-β-actin (Cell Signaling Technology, MA, USA), and then incubated with horseradish peroxidase-conjugated secondary antibodies (Boster, Wuhan, China) for 1 h. Finally, the blots were detected by the enhanced chemiluminescence (ECL) kit (Amersham Biosciences, Piscataway, NJ) [28 (link)].