Irdye 680rd goat anti rabbit lgg h l

SDS-PAGE was performed on SDS-12.5% polyacrylamide gels. Proteins separated by SDS-PAGE were electroblotted onto 0.45-μm reinforced nitrocellulose membranes in transfer buffer (0.5 mM Tris-HCl, 0.2 M glycine, 20% methanol) at 300 mA for 120 min using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). The membranes were blocked in TBST (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween-20) containing 5% nonfat milk at 4°C overnight and then blotted with the primary antibody in TBST buffer for 2 h. After rinsing with TBST five times, the membranes were then transferred to TBST buffer containing 1:10,000-diluted IRDye 800CW goat anti-mouse lgG (H+ L) (1:10,000) (LiCor BioSciences) or IRDye 680RD goat anti-rabbit lgG (H+ L) (1:10,000) (LiCor BioSciences), and thereafter the blots were visualized using an Odyssey infrared imaging system (LiCor BioSciences). Quantification of band intensities by densitometry was performed using the ImageJ software.

Whole cell lysates were prepared at different time points after infection and transfection using RIPA lysis buffer (Sigma-Aldrich, R0278) containing 1 mM phenylmethanesulfonyl fluoride (PMSF; Beyotime, ST506-2) in accordance with the manufacturer’s instructions. The total protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, 23225). Equal amounts of total proteins were analyzed with SDS-PAGE and then transferred onto nitrocellulose membranes (Pall, 66485). After blocking with 5% skim dry milk for 1 h at 37°C, the membranes were incubated with different primary antibodies for 6–8 h at 4°C, and followed by incubation with IRDye 800CW goat anti-mouse lgG (H+ L) (1:10,000) (926–32210; LiCor BioSciences) and IRDye 680RD goat anti-rabbit lgG (H+ L) (1:10,000) (926–68071; LiCor BioSciences) at 37°C for 1 h, and thereafter the blots were visualized using an Odyssey infrared imaging system (LiCor BioSciences). Quantification of band intensities by densitometry was carried out using the Image J software.

Cells were lysed with RIPA lysis buffer (R0278; Sigma–Aldrich) and then centrifuged at 12,000 rpm for 5 min to remove cell debris. Equal amounts of total proteins were analyzed with 12.5% SDS-PAGE and transferred to nitrocellulose membranes (66485; Pall). After blocking with 5% non-fat milk for 1 h at room temperature, the nitrocellulose membranes were incubated with primary antibodies for 6–8 h at 4 °C, followed by incubation with IRDye 800CW goat anti-mouse lgG (H + L) (1:10,000) (926-32210; LiCor BioSciences, Lincoln, NE, USA) or IRDye 680RD goat anti-rabbit lgG (H + L) (1:10,000) (926-68071; LiCor BioSciences) for 45 min in the dark. An Odyssey infrared imaging system (LiCor BioSciences) was used to visualize the blots.