Ab9485

The protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane, followed by blockage with 5% bovine serum albumin (Sigma, B2064) in Tris-buffered saline (Sigma, T5030) containing 0.1% Tween 20 (Sigma, 93773) (TBST). The membranes were then incubated with primary antibodies at 4°C overnight. The primary antibodies included anti-NF-κB p65 (Abcam, ab16502), anti-Nrf2 (Abcam, ab31163), anti-GAPDH (glycer-aldehyde-3-phosphate dehydrogenase) (Abcam, ab9485), and anti-Lamin B1 (Proteintech, 66095-1-Ig) antibodies. After three washes with TBST, the membranes were incubated with HRP-goat-anti-rabbit (Abcam, ab6721) or HRP-goat-anti-mouse (Abcam, ab6789) secondary antibodies at room temperature for 1 h, followed by visualization using an enhanced chemiluminescence (ECL) system (Thermo Scientific, 32132). The images of protein bands were photographed using a ChemiDoc MP device (Bio-Rad, Hercules, CA, USA) and analyzed using ImageJ software.

The samples were lysed using RIPA buffer (Beyotime, China). Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Immobilon P, Millipore, USA). Blots were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h at room temperature. The membranes were incubated with primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies at 37 °C for 1 h. The antibodies included anti-PTEN (Abcam, ab32199, 1:1000), anti-CD9 (Abcam, ab92726, 1:1000), anti-TSG101 (Abcam, ab125011, 1:1000), anti-Annexin II (Abcam, ab178677, 1:1000), anti-calnexin (Abcam, ab92573, 1:1000), anti-GAPDH (Abcam, ab9485, 1:1000), caspase 3 (Proteintech, 19677-1-AP, 1:1000) and horseradish peroxidase-conjugated goat anti-rabbit IgG (Abcam, ab205718, 1:5000). The immunoreactive bands were visualized using an enhanced chemiluminescence reagent (Thermo Fisher Scientific, USA) and imaged by the ChemiDoc XRS Plus luminescent image analyser (Bio-Rad, USA). Densitometric quantification of band intensity from four independent experiments was carried out with Image-Pro Plus 6.0 software, and the relative expression level of the target protein was normalized to the band intensity of GAPDH.

Cellular samples were lysed in reducing Laemmli buffer at 95°C for 10 min. Supernatant or viral stock samples were centrifuged at a relative centrifugal force (RCF) of 18,000 through a 20% sucrose cushion for 1 h at 4°C prior to lysis in reducing Laemmli buffer. Samples were separated on 8 to 16% Mini-Protean TGX precast gels (Bio-Rad) and transferred onto nitrocellulose membranes. Membranes were blocked in milk or Bovine serum albumin (BSA) prior to detection with specific antibodies: 1:1,000 ACE2 rabbit (Abcam; Ab108209), 1:5,000 GAPDH rabbit (Abcam; Ab9485), 1:2,000 anti-GAPDH mouse (Proteintech; 60004-1-Ig), 1:5,000 HSP90 mouse (GeneTex; Gtx109753), 1:50 HIV-1 p24Gag mouse (67 (link)), 1:1,000 spike mouse (GeneTex; Gtx632604), 1:1,000 anti-SARS-CoV-2 N rabbit (GeneTex; GTX135357), 1:1,00 anti-pSTAT1 mouse (BD Transduction Laboratories; 612133), 1:1,000 anti-STAT1 rabbit (Cell Signaling; 9172S), and 1:1,000 anti-viperin mouse (Millipore; MABF106). Proteins were detected using LI-COR and ImageQuant LAS 4000 cameras.