Anti cd36

After the indicated treatments as described in the figure legends, frozen slices or cells were fixed with 4% paraformaldehyde for 15 min at 37 °C and incubated with 0.2% Triton X100 for 15 min at room temperature. After blocking with 3% bovine serum albumin, the slices were incubated with primary antibodies. The following primary antibodies were used: anti-CD36 (#NB600-1423, Novus Biologicals), anti-SELK (#ab121276, Abcam), anti-SEC24 (#15958-1-AP, Proteintech), anti-His-Tag (#12698S, Cell Signaling Technology), anti-Calnexin (#66903-1-Ig, Proteintech), anti-GM130 (#11308-1-AP, Proteintech), anti-Golgin97 (#12640-1-AP, Proteintech), anti-TGN46 (#MA3-063, Invitrogen). After overnight incubation at 4 °C, the slices or cells were then incubated with fluorescence-conjugated secondary antibodies for 1 h. Finally, the slices or cells were incubated with DAPI for 3 min, and then, fluorescence images were captured using a Leica TCS SP8 confocal laser scanning microscope (Leica, Germany) and analyzed using Fiji ImageJ software.

Protein extracts from liver tissue or cells were lysed in RIPA buffer, and cytoplasmic and nuclear proteins were extracted from HepG2 cells or liver tissue using a commercial kit (Beyotime Biotechnology, Wuhan, China). The lysates were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight with primary antibodies at 4°C and then incubated with HRP-conjugated secondary antibodies for 2 hours. Immunoreactive protein was detected using an ECL Advance Western Blotting Detection kit (Amersham Bioscience, Piscataway, US). A quantitative analysis was performed using Image J software (V1.53k). The following primary antibodies were purchased: anti-CD36 (1:2000, Novus, cat# NB400-144), anti-Per1 (1:2000, LifeSpan, cat# LS-C807611), anti-AKT (1:1000, CST, cat# 4691S), anti-p-AKT (1:2000, CST, cat# 4060S), anti-PI3K (1:1000, CST, cat# 4257T), anti-p-PI3K (1:500, GeneTex, cat# GTX132597), anti-FoxO1 (1:1000, CST, cat# 2880S), anti-p-FoxO1 (1:1000, CST, cat# 9461T) anti-G6pase (1:2000, Santa Cruz, cat# sc-167939), anti-PEPCK (1:2000, Santa Cruz, cat# sc-32879) and anti-β-actin (1:5000, Bioss, cat# bs-0061R).

Equal amounts of protein were resolved and immunoblotted with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498), anti-actin (Proteintech Cat# 20536-1-AP, RRID:AB_10700003), anti-NF-κB p65 (Santa Cruz Biotechnology Cat# sc-8008, RRID:AB_628017), anti-Caspase-1 (Proteintech, 22915-1-AP), anti-IL-1β (Proteintech Cat# 16806-1-AP, RRID:AB_10646432), anti-LaminB1 (Proteintech Cat# 12987-1-AP, RRID:AB_2136290), anti-P62 (Abcam Cat# ab109012, RRID:AB_2810880), anti-IP3R1 (GeneTex, GTX133104), anti-phospho-IP3R1 (Thermo Fisher Scientific Cat# PA5-64735, RRID:AB_2662549), and anti-Fyn (Proteintech, 66606-1-lg) primary antibodies. For co-immunoprecipitation (co-IP), equal amounts of lysate proteins were incubated (4°C) with anti-mouse CD36 (Novus Cat# NB600-1423, RRID:AB_789115) overnight before protein G magnetic beads were added (Millipore) (1–3 h). Bound proteins were eluted by boiling for 5 min in SDS sample buffer, and the cleared supernatants were resolved by SDS-PAGE. Detection was performed with the Chemidoc Imaging System (Bio-Rad) and ECL Plus reagent (Amersham). The band intensity was analyzed by densitometry software (ImageJ).

Skeletal muscles were homogenized in a lysis buffer (50 mM Tris-HCl, pH 7.5, containing 1% Triton X-100, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM DTT, 1 mM Na₃VO₄, 10 mM NaF, 10 mM NaPPi, and 10 mM NaMo) and centrifuged at 20,000 × g for 15 min at 4°C. C2C12 myotubes were sonicated in a lysis buffer and centrifuged at 20,000 × g for 15 min at 4°C. The supernatants from homogenates and lysates were subjected to SDS-PAGE and analyzed by Western blot analysis using mouse monoclonal antibodies [anti-MyHC I (clone BA-D5; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), anti-MyHC IIB (clone BF-F3; Developmental Studies Hybridoma Bank, University of Iowa), and anti-β-actin (clone 2D4H5, Proteintech Group, Inc., Chicago, IL) antibodies], and rabbit polyclonal antibodies [anti-HIF-1α (Bethyl Laboratories; Montogomery, TX) and anti-CD36 (Novus Biologicals; Littleton, CO) antibodies]. The immunoreactive proteins were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (BioRad, Hercules, CA) and anti-mouse IgG (BioRad) and reacted with Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA), followed by detection with an LAS4000 imaging system (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, UK).