Anti rack1 antibody

Cells were lysed in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF). Total protein lysates (20 μg) were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and blocked at 4°C for 4 h with 5% nonfat dry milk in Tris-buffered saline (pH 7.5) supplemented with 0.1% Tween 20, followed by overnight incubation with the anti-RACK1 antibody (1 : 1000, Cell Signaling Technology, #5432) or an anti-β-actin antibody (1 : 1000; Abcam, ab8226). An HRP-conjugated secondary antibody (1 : 5000, Thermo Fisher Scientific) was applied for 4 h at 4°C. The intensity for each protein band was corrected by the intensity of the β-actin band and was normalized to facilitate comparisons.

According to our previous study^{32 (link)}, 24 or 48 hours post PRRSV infection, with or without siRNA transfection, the Marc-145 cells were washed with PBS (0.05 M, pH7.4) and fixed with 4% paraformaldehyde (PFA) at room temperature for 10–15 minutes. Then the cells were washed with PBS for three times, permeabilizated with PBS containing 0.3% Triton X-100 for 15 minutes and blocked with PBS containing 1% BSA for 2 hours at 4 °C. The nuclei staining with 5 µg/ml of Hoechst 33342 (Life Technology) was carried out for 20 minutes at room temperature. The cells were subsequently co-incubated with PRRSV antibody against N protein (encoded by ORF7) (VMRD, Cat. #080728-004, mouse origin, 5 µg/ml) and anti-RACK1 antibody (Cell Signaling Technology, Cat. #5432 s, rabbit origin, 2.7 µg/ml) at 4 °C overnight, washed three times with PBS and then co-incubated with Alexa Fluor 488 conjugated goat anti-mouse IgG (H + L) antibody (Proteintech, Cat. #861163) and Alexa Fluor 546 conjugated goat anti-rabbit IgG (H + L) antibody (Thermofisher Scientific, Cat. #11010) at 5 µg/ml for each for 1 h at 37 °C. Staining with Hoechst 33342 (Sigma-Aldrich, Cat. #B2261) at room temperature for 15 minutes was used to visualize the nuclei. After three times PBS wash, the cells were subjected to image analysis by fluorescence microscopy (Olympus).

Glutathione agarose beads (25 µl) coated with either GST or the GST-RACK1 fusion protein (5 µg per sample) were mixed with SUMO or SUMO-BR-C fusion proteins (50 µg per sample) in 1 ml PBS buffer (pH 8.0). Each mixture was incubated with shaking for 8 h at 4°C. The beads were collected by centrifugation and washed three times in PBS buffer and then eluted with 80 µl 50 mM Tris-HCl (pH 8.0) containing 10 mM GSH to isolate the supernatant. The supernatants were mixed with sample loading buffer and separated on SDS-PAGE gels and immunoblotted with anti-His (Sigma; 1∶100) or anti-BR-C antibodies. In addition, we also used the GST-BR-C fusion protein to pull down RACK1 that was prepared from BmN4 cells. The proteins in the supernatants were immunoblotted with anti-RACK1 antibody (Cell signaling technology; 1∶100).