Ab155999

All the proteins were extracted. The cell lysate was run on SDS-PAGE and then transferred to a PVDF membrane (MILLIPORE). The membrane was then blocked with 5% fat-free milk for 2 hours at room temperature. Next, the membrane was probed with a primary antibody at 4°C overnight. The primary antibodies against were SDHC (Abcam, Ab155999, 1 : 10000) and GAPDH (Abcam, Ab8245, 1 : 1000). After washing, the membrane was incubated with appropriate secondary antibodies (Bioswamp) at 4°C overnight as well. The membrane was stained with enhanced chemiluminescence reagent and visualized using the automatic chemiluminescence analyzer (Tanon).

Whole-cell lysates of lung tumours isolated from GEMMs were generated by homogenizing snap-frozen tumour tissues in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% (v/v) Triton X-100, 50 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 2.5 mM pyrophosphate, 1 mM sodium orthovanadate, complete protease inhibitor cocktail). Whole-cell lysates were centrifuged at 2,000g for 5 min and supernatants were transferred to empty tubes. Supernatants were stored at −80 °C until use. Whole-cell lysates of in vitro-cultured cells were generated by homogenizing the cells SDS lysis buffer (100 mM Tris pH 7.5, 100 mM NaCl, 1% SDS, protease inhibitor cocktail) followed by heat inactivation at 90 °C for 10 min. Protein concentration was determined by BCA assay (Thermo Fisher). Lysates were run on 4–12% Bis-Tris gels (Thermo Fisher) to separate the proteins, and then transferred to PVDF membrane. Membranes were stained with Poceau S to confirm transfer efficiency. Membranes were then probed with the following antibodies: SP-C (1:5,000, AB3786 Millipore); GLUT1 (1:2,000, GT11-A, Alpha Diagnostic); NDUFS1 (1:1,000, ab169540, Abcam); O-linked N-acetylglucosamine (1:1,000, ab2739, Abcam); SDHA (1:1,000, 5839, Cell Signaling Technology); SDHC (1:1,000, ab155999, Abcam); actin (1:5,000, A3853, Sigma); tubulin (1:2,500, T9026, Sigma).