Mouse anti actin

Rabbit polyclonal anti-MCDL and anti-MEIK have been described and characterized previously (Liu et al., 2013 (link); Guo et al., 2017 (link)). Rabbit polyclonal anti-NBCn1 was custom-made against an immunogen “ENAKVTRPNMSPEKPVSVTC” by Genscript (Nanjing, CN). Rabbit anti-NHE3 (cat#ab-95299) and mouse anti-α1 (cat#ab-7671) against the α1 subunit of Na⁺-K⁺ pump (aka Na⁺-K⁺-ATPase) were purchased from Abcam (HK, China). Mouse anti-actin was purchased from Beyotime (Cat#AA128, Haimen, Jiangsu, China). HRP-conjugated secondary antibodies were from Beyotime. Alexa Fluor^® 488 AffiniPure goat anti-mouse (cat# 115-545-003) was from Jackson ImmunoResearch (West Grove, PA, United States). Dylight 549 goat anti-rabbit (cat#E032320-01) was from EarthOx (Millbrae, CA, United States).

Twenty micrograms of proteins obtained from lysed cells were denatured and loaded on sodium dodecyl sulfate polyacrylamide gels. Afterward, the proteins were transferred to polyvinylidene difluoride (PVDF) membrane and subjected to Western blotting. Membranes were blocked in Tris-Buffered Saline and Tween20 (TBST) (150 mM NaCl, 10 mM Tris–HCl, pH 7.5 and 0.1% Tween 20) containing 5% (w/v) milk, then incubated with the corresponding primary and secondary antibodies. The primary antibodies used for this experiment were the rabbit anti-ND1, ND5 and A6, mouse anti-CO₁ and CO₂ (Abcam), rabbit anti-ND4 and CYTB (Santa Cruz), and mouse anti-actin (Beyotime). Peroxidase AffiniPure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as a secondary antibody and protein signals were detected using the ECL system (CWBIO).

Engeletin (>99.0% pure, molecular weight: 434.39, CAS: 572-31-6) was obtained from SenBeiJia Biological Technology (China) and was dissolved using dimethyl sulfoxide (DMSO) to prepare a stock solution.
Axitinib (a VEGF receptor (R) inhibitor, Cat No. SC0137-25 mg) and anti-VEGF (Cat No. AV202) were purchased from Beyotime Biotechnology (China). Anti-vasohibin-1 antibody (Cat No.ab176114), anti-Ang-2 (Cat No. ab180820), and anti-Ang-1 (Cat No. ab102015) were from Abcam (UK). Anti-p-Tie2 (Cat No. SAB4503999) and anti-NeuN (Cat No. MEB377) were from Sigma (China). Vasohibin-2 (Cat No. 2923051) was obtained from EMD Millipore (Germany). HRP-labeled goat anti-rabbit IgG (Cat No. A0208), HRP-labeled goat anti-mouse IgG (Cat No. A0216), mouse anti-actin (Cat No. AA128), and BeyoECL Plus (Cat No. P0018FS) were from Beyotime Biotechnology. The 4-0 filament (filament diameter: 0.25 mm; tip diameter: 0.34 mm) was purchased from Beijing Shadong Biology Company (China). RPMI-1640 (Cat No. 11875-093) was from Gibco (USA). Fetal bovine serum (FBS; Cat No.13011-8611) was from Sijiqing (China).