Anti tubulin hfab rhodamine antibody

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).

To obtain nuclear and cytoplasmic extracts from retinas, we performed fractionation with a Nuclear Extract Kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer’s instructions. The protein concentration was determined with Bradford Reagent (BioRad, Hercules, CA, USA) using the Quick Start Bovine Serum Albumin Standard Set (BioRad, Hercules, CA, USA) to obtain a standard curve. A total of 15 μg of protein concentrate was separated by 12% SDS-PAGE at 120 V and transferred onto PVDF membranes (Pall Life Sciences, New York, NY, USA) at 250 mA for 90 min. After transfer, the PVDF membranes were blocked with 3% BSA/TBS buffer for 1.5 h and incubated overnight at +4 °C with estrogen receptor β (ERβ, dilution 1:500, Cloud-Clone Corp., Houston, TX, USA) primary antibody. As a secondary antibody, we used goat anti-rabbit IgG StarBright Blue 700 (BioRad, 1:3000, Hercules, CA, USA) and incubated the membranes for 1 h in the dark at room temperature. We used Anti-tubulin hFAB Rhodamine Antibody (BioRad, 1:3000, Hercules, CA, USA) to normalize protein loading. The membrane signals were detected by multiplexed fluorescence (ChemiDoc MP, BioRad, Hercules, CA, USA). Protein bands were quantified using the Image Lab software.

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).