Imagequant 800 imaging system

Cells were lysed in lysis buffer (20 nM Tris, pH 7.5, 135 mM NaCl, 5% [v/v] glycerol, 50 nM NaF, 0.1% Triton X‐100), as described previously (Dunlop et al., 2014 (link)), with added protease inhibitors. Following sonication, centrifugation and protein quantification, samples were prepared in 4X LDS sample buffer (Invitrogen) with 25 mM dithothreitol (DTT). Lysates were separated by a 4%–12% gradient gel (Invitrogen), transferred onto PVDF membranes, blocked, then incubated with primary antibodies against TSC2 (catalogue #3990), total S6K1 (catalogue #9202), phospho‐S6K1 (T389) (catalogue #9205), pan‐Akt (catalogue #4691), phospho‐Akt (S473) (catalogue #4060) (all Cell Signaling Technology), ALIX (catalogue #sc‐166952), TSG101 (catalogue #sc‐7964), GRP94 (catalogue #sc‐393402) (all Santa Cruz), GAPDH (Novus Biologicals catalogue #1A10), overnight at 4°C. Following washing, secondary antibody incubation and further washes, proteins were visualised using LI‐COR ECL Reagent and a C‐DiGit® Blot Scanner (both LI‐COR Biotechnology) or Amersham ECL reagent and an ImageQuant 800 imaging system (both Cytiva).

The SN was precisely dissected out, and the tissues were incubated with RIPA lysis buffer (CWBIO, China) containing protease inhibitors and lysed for 30 min on ice after being fully ground. After centrifugation at 12,000 rpm for 20 min at 4 °C, the supernatant was removed for determination of the protein concentration by a Bradford assay kit (CWBIO, China). Twenty milligrams of total protein were loaded on a 10% SDS polyacrylamide gel and then transferred onto a 0.45 mm PVDF membrane (300 mV, 90 min). After blocking with 5% nonfat milk at room temperature for 2 h, the membranes were incubated with anti-rabbit TH antibody (Abcam, 1:4000), anti-rabbit CaM antibody (Cell Signaling Technology, 1:1000), anti-rabbit p-CREB antibody (Cell Signaling Technology, 1:1000), anti-rabbit CREB antibody (Cell Signaling Technology, 1:1000), anti-rabbit GHS-R1a antibody (Phoenix Pharmaceuticals, 1:1000), and anti-rabbit GAPDH antibody (Cell Signaling Technology, 1:10,000) overnight at 4 °C. The membranes were then incubated with anti-rabbit secondary antibodies conjugated to horseradish peroxidase at 1:10,000 for 2 h at room temperature. Cross-reactivity was visualized using ECL Western blotting detection reagents and analyzed by scanning densitometry using an Amersham Image Quant 800 imaging system (Cytiva, USA).

The cells were lysed with RIPA (Beyotime) supplemented with protease inhibitor cocktail (Beyotime) and lysates containing equal amounts of protein were boiled and fractionated by 6–10% SDS-PAGE. The primary antibodies included LDHB (11,204–1-AP, Proteintech, PTM-5869, PTM BIO), LDHA (19,987–1-AP, Proteintech), Flag (15,938–1-AP, Proteintech), and β-actin (66,009–1-lg, Proteintech). The signals were detected with Western ECL Substrate (Bio-Rad) using HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Abclonal) and visualized using the AMERSHAM Imagequant 800 imaging System. Experiments were performed at least twice.