B15001

Immunoprecipitation (IP) was performed as previously described [58 (link)]. Briefly, mice liver tissues were homogenized in ice-cold IP lysis buffer (P0013, Beyotime, Shanghai, China) containing a protease inhibitor cocktail (B14001, Bimake, Houston, TX, USA) and phosphatase inhibitor (B15001, Bimake). After preclearing with Protein A/G Plus-Agarose (sc-2003, Santa Cruz), the lysate was incubated with indicated respective antibodies, overnight at 4 °C with gentle rotation, followed by incubation with Protein A/G Plus-Agarose at 4 °C for another 4 h. After washing with an IP lysis buffer three times, the immune complex was boiled with an SDS loading buffer for 10 min. Finally, immunoblotting was performed as described above, except for the second antibody (using a light chain specific antibody).

Assays were performed as described previously (Han et al., 2015 (link); Chen et al., 2017 (link)). In brief, cells were lysed in cold cell lysis buffer (HEPES pH 7.4 50 mmol/L, NaCl 150 mmol/L, Triton X-100 1% and glycerol 10%) supplemented with phosphatase and protease inhibitors (B14011 and B15001, Bimake), sonicated and centrifuged for 15 min at 15,000 rpm at 4 °C. Total protein (20 μg) from each lysate was separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with the indicated antibodies. For immunoprecipitation, control IgG or antibody was incubated overnight at 4 °C with supernatant. Antibodies were purchased as follows: mouse monoclonal anti-HA antibody (MMS-101P), Covance; mouse monoclonal anti-FLAG antibody (F1804) and anti-Tubulin (T5201), Sigma-Aldrich; rabbit polyclonal anti-HA (561) and anti-FLAG (PM020), MBL International; anti-MFN2 (ab56889) and anti-COX4 (ab14744), Abcam; anti-PKM2 (4053), anti-S6K (2708s) and anti-pS6K (9234), Cell Signaling Technology; anti-Myc (SC-40), Santa Cruz. The phospho-S200 MFN2 antibody was generated and purified by AbMax Biotechnology.

The cell or tissue samples were lysed using RIPA Buffer (P0013B, Thermo Fisher Scientific, USA) containing protease and phosphatase inhibitors (78425, Thermo Fisher Scientific, USA; B15001, B15002, Bimake, USA) and protein extraction was performed according to the manufacturer's instructions. Concentration of the protein was determined using the BCA protein assay kit (23225, Thermo Fisher Scientific, USA). Denatured protein diluted to an equal concentration was separated by SDS-PAGE. Subsequently, the protein was transferred to a PVDF membrane at a constant current of 300 mA. After blocked in 5% skim milk for 1 h, the membrane was incubated overnight with the primary antibody: rabbit anti-GFAP antibody (1:1000; 16825-1-AP, Proteintech, USA), mouse anti-HIF-1α antibody (1:200; SC-13515, Santa Cruz, USA), rabbit anti-IBA1 antibody (1:1000; ab178846, Abcam, USA), mouse anti-TH antibody (1:1000; 22941, Immunostar, USA), mouse anti-β-actin antibody (1:1000; SC-47778, Santa Cruz, USA). The next day, after washing, the membrane was incubated with the corresponding secondary antibody for 1 h, and the band of interest was detected using the Odyssey infrared imaging system (Li-Cor, USA). The relative level of protein was quantified using ImageJ software (NIH, Bethesda, MD).

After all treatment, RAW264.7 cells were lysed in RIPA lysis buffer (R0020, Solarbio), containing 1% protease inhibitor cocktail (B14001; Bimake) and 1% phosphatase inhibitor (B15001; Bimake). The protein concentration of lysate was determined by using BCA Assay Kit (P1511-1, Applygen). Twenty micrograms of protein was isolated using 10% SDS/PAGE and electro-transferred to PVDF membranes (IPVH00010; Millipore). The membranes were blocked by TBST containing 5% milk for 2 h at room temperature, and probed with various primary antibodies at 4°C overnight. After being washed with TBST at intervals of 10 min, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. After washing for five times, the immunoreactive bands were visualized using Clarity Western ECL Substrate (170-5060; Bio-Rad). The protein bands were visualized with a Bio-Rad System (Bio-Rad, Hercules, CA, U.S.A.). Actin served as a loading control.

Proteins were extracted from tissues and cells using Radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B Beyotime Biotechnology, Jiangsu, China) with 1× phosphatase inhibitor and protease inhibitor (B15001 and B14001, both Bimake, Houston, TX, USA). After quantification utilizing bicinchoninic acid (BCA)(P0012 Beyotime Biotechnology), protein samples were subjected to SDS–PAGE, followed by electrotransfer to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). 5% skim milk was used to block the membranes for 1 h at room temperature, followed by incubation with the specific primary antibodies overnight at 4 °C. Thereafter, the membrane was washed three times for 10 min each using Tris-buffered saline-Tween 20 (TBST), and then incubated with HRP-conjugated secondary antibodies for 4 h at 4 °C. For immunoprecipitation (IP), IP/Western lysing solution (P0013, Beyotime Biotechnology) was used to lyse the cells and then IP was performed according to the manufacturer’s instructions followed western blotting detection experiments. The quantitative results of western blotting were obtained using ImageJ 1.8.0 software (NIH, Bethesda, MD, USA).

Total protein was isolated using RIPA buffer (C1053, Applygen) supplemented with protease inhibitors (B14001, Bimake) and phosphatase inhibitors (B15001, Bimake) and quantified with a Pierce BCA Protein Assay Kit (23,225, Thermo). Antibodies for immunoblotting were listed in Additional file 1: Table S4.

Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (P0013C, Beyotime) containing a protease and phosphatase inhibitor cocktail (B14011 and B15001, respectively, Bimake) to extract the total protein. Equal amounts of denatured protein samples were separated on 10% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane (IPVH00010, Millipore) for immunoblot analysis. GAPDH was used as an internal control, and the intensity of bands was analyzed using Image J software (version 1.5).