Anti atp1a1

Total protein was obtained using RIPA lysis buffer. Cell membrane, cytoplasm and mitochondrial proteins were obtained using the cell membrane protein and cytoplasmic protein extraction kit (Beyotime) and the cell mitochondrial isolation kit (Beyotime). BCA protein assay Kit (APPLYGEN) was used to quantitate the protein levels. The primary antibody information used is as follows: anti-CLDN10 (1:1000; Abcam, ab52234), anti-Flag (1:1000; CST, 14793S), anti-ATP5O (1:2000; Abcam, ab110276), anti-Acetyl-ATP5O (1:200; Abcam, ab214339), anti-SIRT3 (1:1000; Abcam, ab217319), anti-NDUFS2 (1:5000; Abcam, ab192022), anti-Cleaved-Caspase 3 (1:1000; Affinity, AF7022), anti-E-cadherin (1:10000; Abcam, ab40772), anti-N-cadherin (1:5000, Abcam, ab76011), anti-SDHB (1:5000, Proteintech, China), anti-GAPDH (1:8000, Proteintech, China), anti-ATP1A1 (1:8000, Proteintech, China) and anti-COX Ⅳ (1:8000, Proteintech, China).

A172 cells were treated with 5 μM PGE2 for 24 hours. Cells were lysed with HEPES‐NP40 buffer (20 mM HEPES, 150 mM NaCl, 0.5% NP‐40, 10% Glycerol, 2 mM EDTA, and 1% Protease Inhibitor Cocktail (Sigma, Munich, Germany), pH 7.5) on ice for 45 minutes. After centrifugation at 12 000 g for 15 minutes, the supernatants were mixed with 2× SDS loading buffer and incubated at 95°C for 5 minutes. The protein samples were separated using 8% SDS‐PAGE and transferred to polyvinyldifluoride (PVDF) membranes (Millipore, Burlington, MA, USA). After blocked with 10% nonfat milk in TBST for 1.5 hours at room temperature, the membranes were incubated with primary antibody [Anti‐TrpM7, 1:500 (University of California, Davis); anti‐GAPDH, 1:1000 (Beyotime), Anti‐ATP1A1, 1:800 (Proteintech, Chicago, IL, USA)] in Immunoreaction Enhancer Solution for primary antibody (TOYOBO, Osaka, Japan) overnight at 4°C. The membranes then washed with 0.3‰ TBST for three times and incubated with HRP‐conjugated anti‐mouse IgG (1:1000; Beyotime) for 2 hours at room temperature. Chemiluminescent signals were developed using enhanced chmiluminescence (ECL) reagents (Bio‐Rad) and detected using the ChemiDoc XRS+ System (Bio‐Rad). Image Lab software (Bio‐Rad) was used for quantification of immunoblotting data.