Anti zap

SDS-PAGE and Western blotting were performed as previously described (45 (link)). In brief, at 2 days postinfection, cells were washed with phosphate-buffered saline (PBS) and lysed with coimmunoprecipitation (Co-IP) buffer. The samples were separated on 4% to 12% Bis-Tris gradient acrylamide gels (Invitrogen) and blotted onto polyvinylidene difluoride (PVDF). Blotted membranes were probed with anti-ZAP (GeneTex, catalog no. GTX120134) diluted 1 to 1,000 or with anti-HA tag (catalog no. C29F4; Cell Signaling) diluted 1 to 2,000, anti-KHNYN (Santa Cruz Biotechnology, catalog no. sc-514168) diluted 1 to 50, or anti-TRIM25 (BD Biosciences, catalog no. 610570) or with anti-GAPDH (BioLegend, catalog no. 607902) or anti-beta-actin (abcam, catalog no. ab8227), each diluted 1 to 2,000, or anti-ISG15 (Santa Cruz Biotechnology, catalog no. sc-166755) diluted 1 to 1,000. Proteins were also stained using anti-ACE2 (abcam, catalog no. ab15348) and anti-SARS-CoV-2 Spike (Biozol, catalog no. GTX-GTX632604) primary antibodies, both diluted to 1 to 1,000. Subsequently, blots were stained with IRDye 680RD goat anti-rabbit IgG (H+L) (Li-Cor, catalog no. 926-68071), IRDye 800CW goat anti-mouse IgG (H+L), and IRDye 800CW goat anti-rat IgG (H+L) (Li-Cor, catalog no. 925-32219) secondary antibodies, all diluted 1 to 20,000, and were scanned using a Li-Cor Odyssey reader.

To examine the viral protein levels under the expression of ZAP, HEK293T ZAP KO cells were cotransfected in 12-well plates with 1.25 μg of indicated IMC and 0.25 μg DNA of pCG HA-ZAP IRES BFP vector or empty pCG IRES BFP vector. Two days posttransfection, cells were lysed with coimmunoprecipitation (co-IP) buffer (150 mM NaCl, 50 mM HEPES, 5 mM EDTA, 0.10% NP-40, 0.5 mM sodium orthovanadate, 0.5 mM NaF, protease inhibitor cocktail from Roche), and cell-free virions were purified by centrifugation of cell culture supernatants through a 20% sucrose cushion at 20,800 × g for 90 min at 4°C and lysed in co-IP lysis buffer. Samples were reduced in the presence of β-mercaptoethanol by boiling at 95°C for 10 min. Proteins were separated in 4% to 12% Bis-Tris gradient acrylamide gels (Invitrogen), blotted onto a polyvinylidene difluoride (PVDF) membrane, and incubated with anti-HIV-1 Env (catalog no. 12559; obtained through the NIH AIDS Reagent Program), anti-p24 (catalog no. ab9071; Abcam), anti-HA tag (catalog no. C29F4; Cell Signaling), anti-GAPDH (catalog no. sc-365062; Santa Cruz), and anti-ZAP (catalog no. GTX120134; GeneTex) antibodies. Subsequently, blots were probed with IRDye 680RD goat anti-rabbit IgG(H+L) (catalog no. 926-68071; LI-COR) and IRDye 800CW goat anti-mouse IgG(H+L) (catalog no. 926-32210; LI-COR) Odyssey antibodies and scanned using a Li-Cor Odyssey reader.