Goat anti mouse hrp secondary antibody

Primary antibodies used were as follows: mouse anti-complex V β subunit (CxVβ; for Western Blotting-WB-, 1:1000; for Immunocytochemistry-ICC-, 1:200; Abcam, Cambridge, UK), rabbit anti-peroxisome proliferator-activated receptor γ co-activator 1 α (PGC1-α; for WB, 1:200; for ICC, 1:200; Santa Cruz Biotechnologies, CA, USA), rabbit anti- mtTFA (TFAM; 1:1000, Abcam Cambridge, UK), mouse anti-Mitofusin1 (Mfn1; 1:1000; Abcam, Cambridge, UK), mouse anti-Mitofusin2 (Mfn2; 1:1000; Abcam, Cambridge, UK) mouse anti-mitochondrial dynamin-like GTPase (OPA1; 1:1000; Novus Biologicals, CO, USA), rabbit anti-Dynamin-related protein 1 (Drp1; 1:1000, Cell Signalling Technology, MA, USA) mouse anti-β actin HRP (1:25,000; Abcam, Cambridge, UK), rabbit anti-p62/SQSTM1 (for WB, 1:20,000; for ICC, 1:200; Abcam, Cambridge, UK), rabbit anti-LC3 (for WB, 1:1000; for ICC, 1:200; Novus Biologicals, CO, USA). Secondary antibodies used for WB were as follows: goat anti-rabbit HRP secondary antibody (1:5000; Thermo Fisher Scientific, MA, US), goat anti-mouse HRP secondary antibody (1:5000; Abcam, Cambridge, UK). Secondary antibodies used for ICC were as follows: anti-rabbit (Alexa Fluor, emission at 488 nm; 1:1000; Thermo Fisher Scientific, MA, US) or anti-mouse (Alexa Fluor, emission at 568 nm; 1:1000; Thermo Fisher Scientific, MA, US).

The tagged YN1 mAb clones were analyzed by western blot using anti-Flag staining. Samples were run under reducing and non-reducing conditions on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 4–15% gradient gel (Mini-PROTEAN® TGX™ Gel, Bio-Rad, Hercules, CA). Gels were transferred to PVDF membrane (Millipore, Billerica, MA). Membrane was blocked for 1 h with 3% non-fat dry milk in TBS-T (100 mM Tris, pH 7.5; 150 mM NaCl; 0.1% Tween 20). The membrane was incubated with anti-FLAG M2 antibody (F3165; Sigma-Aldrich, St. Louis, MO), followed by goat anti-mouse HRP secondary antibody (ab6789; Abcam, Cambridge, MA). For chemiluminescence western blot analysis of the modified antibodies, Amersham ECL Western Blotting Detection Reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA) was used.

pVAX1-D3ME or pVAX1 was transfected into Vero cells using Lipofectamine 2000 (Thermo Fisher Scientific, USA). Seventy-two hours after transfection, the supernatant and lysate were collected and fractionated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane according to the manufacturer's recommendation (Millipore, USA). The membrane was blocked with 5% bovine serum albumin for 2 h and incubated with anti-flavivirus E protein monoclonal antibody (1:50, D1-4G2-4-15 hybridoma supernatant, ATCC HB-112) at 4°C overnight, and then probed with goat anti-mouse HRP secondary antibody (1:4,000, abcam, China) for 1 h. Blots were developed with chemiluminescent HRP substrate peroxide solution mix (Millipore, USA) and visualized with Li-Cor Odyssey CLx imaging system.