Anti rabbit igg starbright blue 700

For protein detection, the following commercially available antibodies were used. Primary antibodies: anti-OsTIR1 (MBL, #PD048), anti-mAID (MBL, #M214-3), anti-DHC1 (SantaCruz, #sc-9115), anti-SMC2 (Bethyl, #A300-058A-T), anti-CTCF (Bethyl, #A300-543-T), anti-POLR2A (Abcom, #ab817), anti-BRD4 (GeneTex, #GTX130586), anti-TOP2A (MBL, #M042-3S), anti-alpha-tubulin (MBL, #M175-3), anti-beta-tubulin (SIGMA-Aldrich, #T4026), anti-alpha-tubulin conjugated with rhodamine (Bio-Rad, 12004165). All primary antibodies were used at a 1 in 1000 dilution with TBST containing 5% skim milk. Secondary antibodies: anti-rabbit IgG HRP (GE Healthcare, #NA934), anti-mouse IgG HRP (SantaCruz, #PI-2000), anti-rabbit IgG StarBright Blue 700 (Bio-Rad, #12004161). All secondary antibodies were used at a 1 in 5000 dilution with TBST containing 5% skim milk.

Cell lysates were prepared from either mock- or virus-infected (MOI, 0.01) Vero E6 cells (1.2 × 10⁶ cells/well, 6-well format) after 24 hpi using passive lysis buffer (Promega) based on the manufacturer’s instructions. After centrifugation (12,000 × g) at 4°C for 30 min, proteins were separated with 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega). Then, membranes were incubated at 37°C for 1 h with goat anti-mouse IgG StarBright Blue 520 or anti-rabbit IgG Starbright Blue 700 (Bio-Rad) secondary antibodies. Tubulin was used as a loading control using an anti-tubulin hFAB rhodamine antibody (Bio-Rad). Proteins were detected using a ChemiDoc MP imaging system (Bio-Rad).

Cell lysates were prepared from either mock- or virus-infected (MOI, 0.01) Vero E6 cells (1.2 × 10⁶ cells/well, 6-well format) after 24 hpi using passive lysis buffer (Promega) based on the manufacturer’s instructions. After centrifugation (12,000 × g) at 4°C for 30 min, proteins were separated with 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 h with 5% dried skim milk in 0.1% Tween 20 PBS (T-PBS) and incubated at 4°C overnight with the following specific primary MAbs or polyclonal antibodies (PAbs): N (mouse MAb 1C7C7), mCherry (rabbit Pab; Raybiotech), and Nluc (rabbit Pab, Promega). Then, membranes were incubated at 37°C for 1 h with goat anti-mouse IgG StarBright Blue 520 or anti-rabbit IgG Starbright Blue 700 (Bio-Rad) secondary antibodies. Tubulin was used as a loading control using an anti-tubulin hFAB rhodamine antibody (Bio-Rad). Proteins were detected using a ChemiDoc MP imaging system (Bio-Rad).