Ab110267

Cells were lysed in whole lysis buffer (1% Nonidet P-40, 150 mM NaCl, 50 mM Tris, pH 7.5–8.0, 0.02% sodium azide, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 100 μg/mL PMSF, 1 μg/mL aprotinin), and protein concentrations were determined using the Bio-Rad protein assay. Equal amounts of cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, and then transferred to a 0.45 μm nitrocellulose membrane (Amersham, Sweden). Membranes were blocked with 5% skimmed milk-PBS/0.1%. Tween 20 for an hour prior to an overnight incubation at 4 °C with primary antibody (anti-ABCG2, 1:500 and β-tubulin, 1:200, all diluted in 5% skimmed milk in PBS/0.1% Tween 20). The membrane was then incubated with HRP-conjugated secondary antibody. Following successive washes, membranes were developed using an enhanced ECL detection system. β-tubulin was used as an internal control. Anti-ATP5A antibody (ab110273), abcam and Anti-Cytochrome C Oxidase subunit VIc antibody (ab110267), a cam.

Total protein was extracted using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) and detected utilizing BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). 30 µg of protein per sample was separated by SDS‐PAGE, and then transferred onto PVDF membrane (Gene Molecular Biotech, Inc., Shanghai, China). After obturated with 5% milk for 2 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibodies as follow: COX6C (2 µg mL⁻¹, Abcam, ab110267), DHRS2 (0.1 µg mL⁻¹, Abcam, ab220483), β‐Catenin (1:5000, Abcam, ab32572), Vimentin (1:1000, Abcam, ab92547), E‐cadherin (1:10 000, Abcam, ab40772), Snail (1:1000, Abcam, ab216347), and β‐tubulin (1:1000, Abcam, ab179511). Afterward, the membrane was incubated with HRP‐conjugated goat anti‐rabbit IgG secondary antibodies (1:7500, Abcam, ab205718) or HRP‐conjugated goat anti‐mouse IgG secondary antibodies (1:7500, Abcam, ab205719) for 1 h at room temperature, the expression level was measured with an ECL kit (Roche Diagnostics, Basel, Switzerland) by Western blot imaging system.

Formalin‐fixed, paraffin‐embedded tumor tissues of two additional cases were separately cut into 5‐µm sections and mounted on glass slides. The slides were baked at 65 °C overnight. After deparaffinization and hydration, these slides were boiled in citrate buffer at 100 °C for 15 min. Subsequently, a 3% H2O2 solution was used to block endogenous peroxidase activities for 20 min. To prevent nonspecific antibody binding, the slides were incubated with 5% normal goat serum for 1 h at room temperature. Then these slides were incubated at 4 °C overnight with anti‐COX6C and anti‐DHRS2 primary antibody (Abcam, ab110267 and ab220483), 1:100, which was validated for IHC by the manufacturer on HeLa cells and RT‐4 cells, respectively. Following washes with TBST for three times, the slides were then incubated with HRP‐conjugated goat anti‐rabbit/mouse secondary antibody (GeneTech, GK500705) for 1 h at room temperature. Sections were stained by DAB and then counterstained with hematoxylin according to the manufacturer's instructions.