Ab173305

JQJTT (190210) was provided by Longshunrong Pharmaceutical Factory of Tianjin Zhongxin Pharmaceutical Group Co., Ltd (Tianjin, China). PAL (CAS: 3486-67-7, purity >98%) was provided by Sichuan Vikqi Biotechnology Co., Ltd (Sichuan, China). FGF21 protein was obtained from Northeast Agricultural University. STZ and insulin were purchased from Sigma-Aldrich Co., Ltd (St. Louis, MO, United States). PD173074 was purchased from Selleck Chemicals (Houston, TX, United States). The glucose detection kit was obtained from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Trizol and the cDNA reverse transcription kit were purchased from Thermo Fisher Scientific (Carlsbad, CA, United States). SYBR Green qPCR Mix was provided by Shandong Sikejie Biotechnology Co., Ltd (Shandong, China). Primary antibodies targeting phosphorylated (p)-FGFR1 (ab173305), FRS2 (ab137458), AMPK (ab207442), p-AMPK (ab133448), and β-actin (ab8227) were purchased from Abcam (Cambridge, United Kingdom); those targeting p-FRS2 (YP0805), p-AKT (YT0185), and AKT (YP0006) were obtained from ImmunoWay (California,United States); and that targeting FGFR1 (9740T) was purchased from CST (Boston, MA, United States). Secondary antibodies were obtained from Absin Biotechnology Co., Ltd (Shanghai, China).

HUVECs and HAECs were washed twice with PBS, and lysis with 1 mM phenylmethylsulfonyl fluoride (P7626, Sigma) contained 1X cell lysis buffer (9803, Cell signaling). Quantitative analysis of samples were determined with a Bradford assay dye reagent (500-0006, Bio-Rad). Fifteen μg of sample protein was mixed with 1X loading dye and boiled for 5 min, and electrophoresis on 10% SDS-polyacrylamediume gel. After transferred to the polyvinylidene fluoride membrane (ISEQ00010, Millipore), membranes were blocked in 5% skimmilk contained 1X TBST (WH400028806, 3M) for 1 hr. The membranes were incubated with anti-p-FGFR1 (1:1000, ab173305, abcam), anti-FGFR1 (1:1000, 9740, cell signaling) and anti-GAPDH (1:10000, G8795, Sigma) antibody at 4 °C for overnight. Next, the membranes were washed three times in TBST and incubated with a horseradish peroxidase-conjugated secondary anti-mouse HRP antibody (1:7000, SC-2005, Santa Cruz Biotechnology) and anti-rabbit HRP antibody (1:7000, SC-2030, Santa Cruz Biotechnology) in TBST at room temperature for 90 min. Chemiluminescence were visualized using Amersham ECL Prime Westerm Blotting Reagent (RPN2232SK, GE Healthcare Life Sciences) and exposed to X-ray film. Quantification of blotting intensity were performed using Quantity One (Bio-Rad) program.