Ab171941

qRT-PCR was performed as previously described^{22 (link)}. The primer pairs are listed in Table S1. Western blot analysis was performed as described previously^{23 (link)}. The primary antibodies included anti-LC3 (1:1000, NB100-2220, Novus Biologicals, Colorado, USA), anti-FGF21 (1:1000, ab171941, Abcam, Cambridge, UK), anti-P62 (1:1000, ab56416, Abcam, Cambridge, UK), anti-Ki-67 (1:1000, ab16667, Abcam, Cambridge, UK), anti-LAMP2 (1:500, ab13524, Abcam, Cambridge, UK), anti-BCL-2 (1:1000, #15071T, Cell Signaling Technology, Massachusetts, USA), anti-Akt kinase (Akt; 1:1000, #9272s, Cell Signaling Technology, Massachusetts, USA), phospho-Akt (Ser 473; 1:1000, #4060s, Cell Signaling Technology, Massachusetts, USA), anti-mTOR (1:1000, #2972s, Cell Signaling Technology, Massachusetts, USA), phospho-mTOR (1:1000, #2971s, Cell Signaling Technology, Massachusetts, USA), anti-p70S6K (1:1000, #2708s, Cell Signaling Technology, Massachusetts, USA), phospho-p70S6K (Thr389; 1:1000, #9205s, Cell Signaling Technology, Massachusetts, USA), and anti-β-actin (1:1000, TA-09, ZSGB-BIO, Beijing, China).

Frozen GAS and C2C12 cells were homogenized in RIPA buffer (R0010, Solarbio) containing 1 mM PMSF (P0010, Solarbio), and protease inhibitor cocktail (539133, Merck, Rahway, NJ, USA). The protein concentration was measured using a BCA protein assay kit (P0012, Beyotime, Shanghai, China). Equivalent proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride (PVDF) membranes (ISEQ00010, Merck). After blocking the membrane with 5% non-fat dry milk for 1.5 h, primary antibodies were proceeded overnight at 4 °C and included rabbit anti-Fgf21 (ab171941, Abcam), rabbit anti-MyHC I (22280-1-AP, Proteintech), rabbit anti-MyHC IIa (ab124937, Abcam), rabbit anti-MyHC IIb (20140-1-AP, Proteintech), and mouse anti-MyHC IIx (67299-1-Ig, Proteintech), rabbit anti-TGF-β1 (bs-0086R, Bioss, Beijing, China), rabbit anti-Smad2/3 (PA5-99539, Invitrogen), rabbit anti-p-p38 MAPK (8690, CST, MA, USA), mouse anti-Tubulin (T6199, Sigma-Aldrich, MO, USA), and mouse anti-β-actin (4967, CST). HRP-conjugated goat anti-mouse (CW0102S, CWBIO, Taizhou, China) or anti-rabbit (CW0156S, CWBIO) secondary antibodies were incubated at room temperature for 1 h. ECL (PEOO10, Solarbio) was used for enhanced chemiluminescence detection, according to the manufacturer’s instructions.

Protein lysates were prepared from the liver tissue using the RIPA lysis buffer (Beyotime, Shanghai, China), and the protein concentration was measured using the BCA protein detection kit. Then, 20 μg protein samples were denatured at 95 °C for 5 min. After SDS-PAGE electrophoresis, the proteins were transferred onto a PVDF membrane at 200 mA for 90 min. The membrane was blocked at room temperature with a quick blocking buffer (SunBioo, Beijing, China) for 10 min. Subsequently, the membranes were incubated with Anti-ACSL1 (1:3000, 13989-1-AP, Proteintech, Chicago, IL, USA), Anti-CPT1A (1:2000, 15184-1-AP, Proteintech, Chicago, IL, USA), Anti-FGF21 (1:1000, ab171941, Abcam, Cambridge, UK), and Anti-GAPDH (1:1000, ab56416, Abcam, Cambridge, UK) at 4 °C overnight. After washing, horseradish peroxidase-conjugated anti-rabbit antibodies were added for further incubation, and then, the signal was developed using a chemiluminescent imaging system and quantified using ImageJ software. Photoshop CC 2019 was used to crop images from unprocessed images.

Mouse liver tissues were homogenized in RIPA buffer with protease and phosphatase inhibitors (Bimake, Houston, USA). The protein concentration was determined using a BCA Protein Quantitative Assay Kit (Shenergy Biocolor Bioscience & Technology Co., Shanghai, China). Total protein was mixed with SDS loading buffer and subjected to SDS-PAGE on a 10% gel. Proteins were electrotransferred onto PVDF membranes (Millipore) and the blots were probed with the following primary antibodies overnight at 4 °C: anti-FASN (cst3180, Cell Signaling Technology, USA), anti-SREBP1C (ab3259, Abcam, USA), anti-SCD1 (ab19862, Abcam), anti-CPT1α (ab176320, Abcam), anti-MTP (sc-135994, Santa Cruz Biotechnology, USA), anti-CD36 (18836-i-ap, Proteintech, China), anti-FGF21 (ab171941, Abcam), anti-BIP (11587-1-ap, Proteintech), anti-p-IRE (ab48187, Abcam), anti-IRE (ab37073, Abcam), anti-eIF2α (cst9722, Cell Signaling Technology), anti-p-eIF2α (cst3597, Cell Signaling Technology), anti-ATF4 (10835-1-ap, Proteintech), anti-ATF6 (ab122897, Abcam), anti-p-PERK (sc-32577, Santa Cruz Biotechnology), anti-PERK (ab65142, Abcam) and anti-GAPDH (60,004–1, Proteintech). Appropriate secondary antibodies conjugated to horseradish peroxidase (Amersham) were diluted 1:5000 used. The bound primary antibodies were visualized using the Alpha Q detection system.

For Western blot analyses in mouse tissues, samples were homogenized and sonicated in T‐PER^® buffer (Thermo Scientific) with protease inhibitor cocktail (Pierce). For Western blot analyses in brain mitochondria, the pellets containing the mitochondrial fraction were re‐suspended in RIPA buffer with protease inhibitor cocktail. Protein band intensity was normalized to VDAC1 (mitochondrial proteins), and the data were expressed in terms of percent relative to wild‐type mice or control cells (Luna‐Sanchez et al, 2015, 2017). The following primary antibodies were used: anti‐SQRDL (Proteintech, 17256‐1‐AP, dilution 1:500), anti‐PDSS2 (Proteintech, 13544‐1‐AP, dilution 1:500), anti‐COQ2 (Origene, TA341982, dilution 1:2,500), anti‐COQ4 (Proteintech, 16654‐1‐AP, dilution 1:2,000), anti‐COQ5 (Proteintech, 17453‐1‐AP, dilution 1:1,000), anti‐COQ6 (Proteintech, 12481‐1‐AP, dilution 1:500), anti‐COQ7 (Proteintech, 15083‐1‐AP, dilution 1:1,000), anti‐COQ8A (Proteintech, 15528‐1‐AP, dilution 1:2,500), anti‐S6R (Cell Signaling, 2217, dilution 1:1,000), anti‐S6RP (Cell Signaling, 2211, dilution 1:1,000), anti‐FGF21 (Abcam, ab171941, dilution 1:1,000), anti‐VDAC1 (Abcam, ab14734, dilution 1:5,000), anti‐TOM20 (Proteintech, 11802‐1‐AP, dilution 1:5,000), and anti‐GAPDH (Santacruz, sc‐166574, dilution 1:200).