Ecl plus kit

Total proteins were extracted using RIPA buffer (Solarbio, Beijing, China) containing 1% PMSF (Solarbio, Beijing, China). Protein concentrations were determined using the BCA protein concentration assay kit (Solarbio, Beijing, China). Protein samples (10–20 µg) were diluted to the same volume with Loading Buffer and then separated by 5% SDS-PAGE before transferring to PVDF membranes (Millipore, Billerica, MA, US). The membranes were blocked in 5% skim milk (Sangon, Shanghai, China) for 1 h and then incubated with the specific primary antibodies overnight at 4 °C. The primary antibodies are as follows: KHDRBS3 (Santa Cruz Biotechnology, sc-374,461, Shanghai, China; 1:500); c-Myc (ABclonal, A1309, Shanghai, China; 1:500); GLUT1 (Affbiotech, AF5462, Changzhou, China; 1:500); LDHA (ABclonal, A1146, Shanghai, China; 1:1000); 14-3-3ζ (Affbiotech, AF6356, Changzhou, China; 1:1000); GAPDH (Proteintech, 60004-1-Ig, Wuhan China; 1:10000). After incubation with HRP-conjugated secondary antibodies (Solarbio, Beijing, China), the bands were visualized using ECL Plus kit (Solarbio, Beijing, China).

Total proteins were extracted from MAC-T cells using RIPA lysis buffer supplemented with PMSF (Solarbio, Beijing, China) and quantified with a BCA protein assay (Solarbio). Equal amounts of protein were separated on 10% SDS-PAGE gels. Primary antibodies against FADS2 (catalog #PA5-25285) were purchased from Thermo Fisher Scientific. Antibodies against β-actin (catalog #SC-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary HRP-conjugated goat anti-rabbit IgG antibody (catalog #31210) was purchased from Thermo Fisher Scientific. Signals were visualized with the ECL Plus kit (Solarbio) and imaged under the ChemiDoc XRS System (Bio-Rad, Hercules, CA, USA).