Anti ghpdh

Tumor tissues and cells were washed with PBS and underwent lysis with RIPA buffer (Pierce) supplemented with protease inhibitors (Boster Biological Technology, China). Protein quantitation utilized the BCA assay kit (Beyotime Biotechnology). Equal amounts of total protein were resolved by SDS-PAGE and underwent transfer onto polyvinylidene fluoride membranes (Millipore, USA). Western blot membranes underwent overnight incubation at 4 °C with anti-FAM126A (1:1000; Sigma, #84668), anti-ENO1 (1:1000; Proteintech, #11204), anti-GHPDH (1:1000; Proteintech, #60004), anti‑E‑cadherin (1:1000; Proteintech, #20874), anti-N-cadherin (1:1000; Proteintech, #22018), anti‑vimentin (1:1,000; Proteintech, #10366), anti-snail (1:1000; Proteintech, #13099), anti-cyclin D1 (1:1000; Cell Signaling Technology [CST], #55506), anti-cyclin E1 (1:1000; CST, #4136), anti-CDK 2 (1:1000; CST, #2561), anti-CDK4 (1:1,000; CST, #12790), anti-AKT (1:1000; CST, #4691), anti-p-AKT (1:2000; CST, #4060), anti-PI3K (1:1000; CST, #4249), anti-p-PI3K (1:1000; CST, #17366), anti-Ki-67(1:800; CST, #9449) and anti-PCNA (1:1000; CST, #2561) primary antibodies. This was followed by incubation with HRP-linked anti-rabbit IgG (Boster, #BA1041). Enhanced chemiluminescence reagent (Proteintech, #7003) was used to detect immunoreactive signals.

Duodenum was harvested from the mice that were injected daily with saline or PF (1 mg/kg) for 7 consecutive days and homogenated with RIPA buffer (Thermo Fisher Scientific) containing 1 mM PMSF. After centrifugation at 16,000 g for 20 min, the supernatant was collected and quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific). Seventy-five micrograms of proteins were separated using 10% SDS-PAGE and transferred to PVDF membranes (PerkinElmer). The membranes were blocked using 5 % (w/v) skim milk for 1 h at room temperature, followed by incubation overnight at 4°C with the primary antibody anti-MBOAT4 (1:1,000; Bioss; catalog# bs-13355R) or anti-GHS-R1 (H–80) (1:1,000; catalog# sc-20748; Santa Cruz Biotechnology), and then incubated for 1h at room temperature with the secondary antibody peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L), or and goat anti-mouse IgG (H+L) (1:5000, ZSGB-Bio, Beijing, China). The protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (anti-GHPDH, 1:5,000; catalog# 60004-1-Ig; Proteintech Group, Rosemont, IL, United States) on the same gel.

Anti-FAM63B (1:500; ThermoFisher, # 62318), Anti-ACTN4 (1:5,000; Proteintech, #66628), Anti-GHPDH (1:1,000; Proteintech, #60004), anti-E-calmodulin (1:1,000; Proteintech, #20874), anti-N-calmodulin (1:1000; Proteintech, #22018), anti-wavoprotein (1:1,000; Proteintech, #10366), anti-snail (1:1000; Proteintech, #13099), anti-cyclin D1 (1:1,000; Cell Signaling Technology [CST], #55506), anti-cyclin E1 (1:1,000; CST, #4136), anti-CDK 2 (1:1,000; CST, #2561), anti-CDK4 (1:1,000; CST, #12790), anti-AKT (1:1,000; CST, #4691), anti-p-AKT (1:2,000; CST, #4060), anti-PI3K (1:1,000; CST, #4249), anti-p-PI3K (1:1,000; CST, #17366), anti-mTOR (1:1,000; CST, #2972), anti-p-mTOR (1: 1000; CST, #2971), HRP-goat anti-rabbit IgG (Boster, #BA1055), HRP-goat anti-mouse IgG (Boster, #BA1050), Anti-PCNA (1:1,000; Proteintech, # 10205), Anti-Ki67 (1:1,000; Proteintech, #28074), HA-Ubiquitin plasmid (Sangon Biotech, China), CHX (Melun Biologics, China), MG132 (MCE, USA), MINDY2 small interfering RNA (RiboBio, China), protease inhibitor (Boster Biological Technology, China), and enhanced chemiluminescence reagent (Proteintech, #7003).