Anti flag tag

Mitochondrial fractions were isolated from WT and OSGEPL1 KO HEK293T cells (1 × 10⁷ cells) using the Mitochondria Isolation Kit (Miltenyi Biotec. K.K.) and subjected to western blotting to measure steady-state levels of protein components in respiratory chain complexes using the Total OXPHOS Rodent WB Antibody Cocktail (an antibody mixture targeting ATP5A, UQCRC2, MTCO1, SDHB, and NDUF88; ab110413, Abcam), an anti-ND5 antibody (ab92624, Abcam), and an anti-ND2 antibody (19704-1-AP, Proteintech). Other antibodies used in this study were as follows: anti-OSGEPL1 (25694-1-AP, Proteintech), anti-GAPDH (6C5, Santa Cruz Biotech), anti-FLAG-tag (1E6, Wako), HRP-conjugated donkey anti-mouse/rabbit IgG (715-035-150/715-035-152, Jackson ImmunoResearch), anti-FLAG M2 affinity gel (A2220, Sigma), and Alexa Fluor 488-conjugated goat anti-mouse IgG (A-11001, ThermoFisher). To monitor YRDC subcellular localization using the anti-FLAG-tag antibody, mitochondrial fractions were carefully washed with 2 mg/mL digitonin (Sigma) to remove the outer membrane with cytoplasmic components.

HEK293T cells were seeded in 6-well plates and transfected with the indicated plasmids. After 24-h incubation, cells were scraped and resuspended in Pierce IP Lysis Buffer (Thermo Fisher Scientific, USA) supplemented with protease inhibitor cocktail (Roche). Cell debris was removed by centrifugation (16,000 ×g, 4°C, 15 min). Antibodies were added and incubated with 50 μl protein A magnetic beads (SureBeads, Bio-Rad, USA) with rotation. The beads were washed extensively with PBS supplemented with 0.1% Tween 20. Beads probed with appropriate antibodies were incubated with 300 μg proteins 4°C with rotation. Proteins were analyzed by immunoblot analysis. Protein samples were probed with the following primary antibodies: anti-FLAG tag (1:2000; Fujifilm Wako Pure Chemical Corporation, Japan), anti-MYC tag (1:2000; Abcam, UK)

RIPA buffer (Sigma-Aldrich) plus a protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland) was used to lyse cells as previously described [18 (link)]. Proteins were loaded onto SDS-PAGE gels, transferred onto polyvinylidene difluoride membranes, and blocked with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween-20 (TBS-T) for 1 h at room temperature. The membranes were incubated overnight with primary antibodies at 4 °C. The following antibodies were used; Anti-FLAG tag (Wako), Anti-MYC tag (Abcam), Anti-GFP (Santa Cruz), Anti-Tubulin or β-actin antibodies (Abgent). Anti-pSTAT1/STAT1, Anti-HA tag, Anti-HIS tag, Anti-MYC tag, Anti-MDA5, Anti-RIG-I, Anti-pTBK1/TBK1, Anti-pIRF3/IRF3, Anti-MxA, Anti-OAS1, and Anti-RNaseL antibodies were from Cell Signaling Technology. Anti-SARS-CoV/SARS-CoV-2 Nucleocapsid Antibody (Sino Biological), while Anti-IFV NP antibody and Anti-ZIKV E antibody were purchased from GeneTex. The blots were washed with TBST for three times and incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). Proteins were visualized with ECL Western Blotting Substrate (Thermo Fisher Scientific, Waltham, MA, USA). Densitometry to quantify immunoblot bands was performed using ImageJ software.