Nonidet p40 substitute

Total cell extract was prepared with 1 × RIPA buffer [50 mmol/L-Tris-HCl buffer (pH 7.6), 150 mmol/L-NaCl, 1 %-Nonidet P40 substitute (w/v), 0.5 %-sodium deoxycholate (w/v), protease inhibitor cocktail, 0.1 %-SDS (w/v)] (FUJIFILM Wako Pure Chemical). The samples were separated on 10% SuperSep Ace gels (Wako) and transferred to PVDF membranes (Merck). Nonspecific binding was blocked at room temperature (15–25 °C) for 15 min with Blocking One-P (Nacalai Tesque, Kyoto, Japan). The membranes were incubated overnight at 4 °C with the following antibodies: anti-KIAA1199 (HYBID) rabbit polyclonal antibody (pAb) (1:1000) (Sigma Aldrich, MO, USA), anti-TMEM2 rabbit pAb (1:500) (Sigma Aldrich), anti-FLAG M2 mouse mAb (1:2000) (Sigma Aldrich) and anti-GAPDH mouse mAb (1:2000) (Santa Cruz Biotechnology). The primary antibodies were detected using HRP-conjugated secondary antibodies (GE Healthcare). Bands were detected via ImmunoStar LD (FUJIFILM Wako Pure Chemical) using an Amersham Imager 680 (GE Healthcare Bioscience, NJ, USA).

For MARV, HEK293F cells grown in 10 ml of medium (3.0 × 10⁶ cells/ml) were transfected with 6 µg of pCAGGS-NP (1–395) using Polyethyleneimine MAX (Polysciences, Warrington, PA, USA). Three days post-transfection, the cells were collected and lysed with 0.1% Nonidet P-40 substitute (Wako, Osaka, Japan) in Tris-HCl buffer (10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA) including a cOmplete Protease Inhibitor (Roche, Basel, Switzerland) and 10 mM Ribonucleoside-Vanadyl Complex (NEB, Ipswich, MA, USA) on ice. The lysate was centrifuged at 10,000 g at 4 °C for 10 min to remove insoluble substances, and the supernatant was subjected to a discontinuous CsCl gradient ultracentrifugation at 246,100 g at 4 °C for 2 h. Then, fractions containing the NP–RNA complex were collected and ultracentrifuged at 246,100 g at 4 °C for 15 min. The pellet was suspended in Tris-HCl buffer.
For EBOV, HEK293T cells grown in 10 ml of medium (3.0 × 10⁵ cells/ml) were transfected with 10 µg of pCAGGS-NP (1–450) using TransIT-293 Reagent (Takara, Shiga, Japan). Three days post-transfection, NP–RNA complexes were purified as described above and suspended in Tris-HCl buffer.