β actin

RIPA buffer, which contained a protease inhibitor (Cell Signaling Technology, Danvers, MA, USA), was applied for cell lysis. The protein concentrations were determined using a BCA protein assay kit (KeyGEN BioTECH, Nanjing, China). SDS-PAGE (8–10%) was used to separate the proteins, which were then transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). The primary antibodies included HIF-2α (1:500, ab109616, Abcam, Cambridge, UK), c-Myc (1:500, #13987, Cell Signaling Technology), GOT1 (1:1000, #14886-1-AP, Proteintech, Rosemont, IL, USA), and β-actin (1:5000, #AP0060, Bioworld, Bloomington, MN, USA). Enhanced chemiluminescence (Merck Millipore) was used to evaluate the signal, and analysis was performed using the ImagePro Plus software (Media Cybernetics, Rockville, MD, USA). Each target gene protein was semi-quantitatively estimated, compared with β-actin as an internal loading control.

Total protein, membrane protein, cytoplasmic protein, and nucleoprotein were extracted from RA FLS and HEK293T cells by ProteoPrep® Total Extraction Sample Kit (Sigma-Aldrich), ProteoPrep® membrane Extraction Kit (Sigma-Aldrich), and NE-PER™Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific), respectively. Protein in culture medium (100 ml) was pretreated by freeze-drying method and then dissolved in an appropriate amount of double distilled water. After quantification with Pierce BCA Protein Assay Kit (Thermo Fisher Scientific), 25 μg protein samples were separated by SDS-PAGE and then electrotransfered onto polyvinylidene difluoride membranes. After blocked in 5% skim milk for 2 h, the membranes were incubated with antibodies of KIAA1199 (#ab98947, Abcam), HYAL2 (#ab126099, Abcam), CD44 (#ab157107, Abcam), AXNA1 (#ab88865, Abcam), PI3K (#4249, CST), total-Akt (#2920, CST), p-Akt (Ser473) (#4060, CST), total-STAT3 (#4904, CST), p-STAT3 (Tyr705) (#9131, CST), p-P65 (Ser536) (#3036, CST), and then with matching HRP-conjugated secondary antibodies. Protein bands were visualized using ECL substrate (#35055, Thermo Fisher Scientific). β-Actin (#AP0060, Bioworld) and Lamin B1 (#12987-1-AP, Proteintech) were used as the internal reference of cytoplasmic protein and nucleoprotein, respectively. The intensity of protein bands were analyzed by Image J (v1.8.0).

PBMCs (1 x 10⁷ cells) were lysed by 200 μl RIPA lysis buffer (CWBIO, Beijing) supplement with protease inhibitor cocktail (CWBIO, Beijing) and phosphatase inhibitor cocktail (CWBIO, Beijing). After lysis at 4°C for 15 min, the proteins were centrifuged at 8000 x g for 15 min. The protein concentration in the supernatant of cell extracts was determined using a bicinchoninic acid protein assay kit (Beyotime, Shanghai). Then, proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime, Guangdong) and transferred to polyvinylidene difluoride membranes (Millipore, American). The membranes were blocked with 5% no-fat powdered milk (Sangon, Shanghai) in tris-buffered saline (Solarbio, Beijing) with 1% tween (Solarbio, Beijing) for 2 h at room temperature. The membrane was probed with diluted primary antibodies CITED2 (Abclonal, 1:1,000), VEGF (Bioss, 1:1,000), and β-actin (Bioworld, 1:10,000) overnight at 4°C. On the next day, the membrane was re-probed with a secondary antibody, goat anti-rabbit antibody IgG (CWBIO, 1:20,000), labeled by enhanced chemiluminescence hypersensitive luminescent solution (Millipore, American) for 2 h at room temperature, and quantified by densitometry.

Total protein was extracted from 40 mg frozen gastrocnemius muscle samples as previously described [41 (link)]. Protein concentration was measured with a Pierce BCA Protein Assay kit (23225; Thermo Scientific). Antibodies and their sources are: Adiponectin (BS6961, Bioworld), AdipoR1 (BS6797, Bioworld), AdipoR2 (14361-1-AP, Proteintech), AMPKα112 (BS1009, Bioworld), p-AMPKα1/2 (sc-33524, Santa Cruz), PGC-1α (sc-13067, Santa Cruz), UCP3 (BS2849, Bioworld), UCP2 (BS2917, Bioworld), GPR43 (sc-32906, Santa Cruz), GPR41 (sc-98332, Santa Cruz), CPT-1b (sc-20670, Santa Cruz), COX4 (MB0102, Bioworld), HDAC1 (BS6485, Bioworld), GAPDH (AP0063, Bioworld), β-actin (AP0063, Bioworld). The phosphorylated AMPK was normalized by the total AMPK.

The 4^th mammary glands excised from mice were snap frozen in liquid nitrogen, then lysed in cold RIPA buffer (1 × PBS, 1% NP-40, 0.5% sodium dexoycholate, 0.1% SDS, protease inhibitor cocktail tablet [Roche] and 1 mM PMSF [Beyotime Biotechnology]). Lysates were incubated on ice for 20 minutes with frequent vortexing and cleared twice by centrifugation (13,200 rpm,10 minutes, 4 °C). Protein was subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA). Membranes were blocked for 60 minutes at room temperature in 5% non-fat milk/Tris-buffered saline/0.1% Tween (TBST). After being washed with TBST, the blots were incubated in primary antibodies for 3 h, which were PS6 (1:2000, CST), S6 (1:2000, CST), TSC1 (1:1000, Proteintech), PDK1(1:1000, ABclonal), phospho-PDK1 (1:1000, Bioworld) phospho-AKT308 (1:1000, Bioworld), phospho-AKT473 (1:1000, CST), AKT1 (1:1000, CST) and β-actin (1:1000, Bioworld). Antibody detection was performed according to the manufacturer’s instructions with ECL Plus Western Blotting Detection System (Genstar, Beijing, China) and developed on film.

Liver tissues and HSCs were lysed in RIPA buffer for protein extraction by using centrifugal separation. Then we collected upper supernatant. After extraction, protein concentration was estimated by a BCA protein assay kit (Boster, China). Proteins extracted were loaded on to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes (Millipore, Bedford, MA, United States). After blocked with 5% non-fat milk in TBST, the PVDF membranes were washed with TBST buffer at least for three times and cultured with special primary antibodies at 4°C overnight. Then the PVDF membranes were washed for three times with TBST buffer. Next, the membranes were incubated with corresponding secondary antibody for 1 h. Immunoreactive bands were detected with ECL-chemiluminescent kit (ECL-plus, Thermo Fisher Scientific). Image J software was used to quantify and analyze intensities of the bands. β-actin was developed as a loading control to verify the equal loading of proteins. Primary antibodies were as follows: ZEB2 (Santa Cruz Biotechnology, United States); TRAIL, Col. I, Caspase-3 (Bioss, Beijing, China), α-SMA (Sigma, United States), β-actin (Bioworld, United States).

The Western blot analysis was carried out as per standard protocols on 10% SDS/PAGE gels and then transferred to nitrocellulose membranes. The membranes were blocked in TBST with 0.1% Tween-20 and 5% non-fat dry milk for 2 h and then incubated with primary antibodies against Mtfmt (1:1000, cat. no.: PAH614Mu01, Cloud-Clone Corp, Houston, TX USA), p-IkBα (1:1000, cat. no.: 2859S, Cell Signaling, Danvers, MA, USA), IkBα (1:1000, cat. no.: 9242, Cell Signaling, Danvers, MA, USA), IL-1β (1:1000, cat. no.: ab254360, abcam, Cambridge, UK), SDHA (1:1000, cat. no.: 14865-1-AP, proteintech, Rosemont, IL, USA), VDAC (1:1000, cat. no.: 10866-1-AP, proteintech, Rosemont, IL, USA), ND6 (1:1000, cat. no.: BS1632, Bioworld, Nanjing, China), COX1 (1:1000, cat. no.: BS70809, Bioworld, Nanjing, China), β-actin (1:20,000, cat. no.: AC026, Abclonal, Wuhan, China), and GAPDH (1:1000, cat. no.: MB001H, Bioworld, Nanjing, China).