Fgfr1

Protein samples (20 μg) were separated using a 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gel and transferred to polyvinylidene fluoride (PVDF) membranes by using a Bio-Rad Mini-Protein electro-transfer system (Bio-Rad Laboratories, Inc., CA, USA). Next, the PVDF membranes were blocked in 5% nonfat milk in Tris-buffered saline with Tween 20 for 1 h. After blocking, the membranes were incubated at 4 °C overnight with primary antibodies against FGFR1 (#9740; 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), PARP (#9532; 1:1000, Cell Signaling Technology), cleaved PARP (#5625; 1:1000, Cell Signaling Technology), caspase-9 (#9508; 1:1000, Cell Signaling Technology), caspase-3 (#9668; 1:1000, Cell Signaling Technology), Bcl-xL (#2764; 1:1000, Cell Signaling Technology), and β-actin (sc-69879; 1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA). All the antibodies used are listed in Supplementary Table S1. The membranes were then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h and washed carefully with PBS three times. Next, protein band detection was performed using the enhanced chemiluminescence detection system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The protein bands were quantified using ImageJ (https://imagej.nih.gov/ij/).

Indicated cells were lysed with RIPA buffer. Protein lysates were electrophoresed through SDS polyacrylamide gel, followed by transferring to PVDF membranes (Millipore, USA). The membranes were blocked with non-fat dry milk at room temperature and then incubated with primary antibodies at 4°Covernight. The primary antibodies included: FGFR1, PDHK1, P-PDHK1, LDHA, P-LDHA, MMP-1 and β-actin (Cell Signaling Technology). Membranes were then washed and incubated with secondary antibodies. Proteins were visualized by the electrochemiluminescence (ECL) western blot substrate detection (Pierce).

Liver tissues were homogenized and determined as described previously (Wang et al., 2019 (link)). The blots were incubated with primary antibodies: cleaved caspase-3, Bax, Bcl-2, ATF6, GRP78, Nrf2, SOD2, collagen IA1, ATG5, p62 and LC3 (Abcam, USA), HO-1 (Santa Cruz Biotechnology, CA, USA), CHOP, P-IRE1α, P-eIF2α, P-PERK, PERK, FGFR1, P-AMPK and AMPK (Cell Signaling Technology, Danvers, MA, US), and α-SMA, TGF-β, and GAPDH (Proteintech, China) followed by incubation with their corresponding secondary antibodies: anti-rabbit and anti-mouse (Proteintech, China).

The dissected liver tissues were stored in liquid nitrogen until analyses (n = 6 per group). Otherwise, insulin (0 and 10 U/kg) was intraperitoneally administrated 15 min prior to tissue collection in certain rats (n = 6 per group). The tissues were homogenized in tissue-protein extraction reagent (T-PER, Pierce Biotechnology, Rockford, IL, USA). After sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) separation and transfer, the polyvinylidene fluoride (PVDF) membranes reacted with primary antibodies, horseradish peroxidase-labeled IgG, subsequently followed by Enhanced Chemiluminescence (ECL) detection reagents. The intensity of recognized protein was quantified through densitometry. The primary antibodies recognized included GLP-1R (1:1000, sc-390774), Klotho (1:500, sc-515939), Akt (1:2000, sc-8312), phospho-Akt (1:1000, sc-7985-R), ERK1/2 (1:2000, sc-514302), phospho-ERK1/2 (1:1000, sc-7383), PERK (1:1000, sc-9479), phospho-PERK (1:1000, sc-32577), FGF-21 (1:500, sc-16842), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2000, sc-32233) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), FGFR1 (1:1000, #9740), phospho-FGFR1 (1:1000, #3471) (Cell Signaling, Danvers, MA, USA).