Lysis buffer

All SARS-CoV-2 infection experiments were performed at the URMC BSL3 laboratory following the approved standard operating procedures.
Infection of BST2 stable cell lines. 10⁶ HEK293T-ACE2 and A549-ACE2 cells stably expressing pQCXIP or pQCXIP-BST2 were seeded in 6-well plates. Twenty-four hours later, cells were infected with different SARS-CoV-2 strains at MOI = 0.1 or 1 (see Table 1 for virus strains). As controls, we included untreated cells (NT) and mock-infected cells, which consisted of lentiviral-like particles harboring GFP. Twenty-four hours post-infection, supernatants were collected to assess virion production and infectivity, and cells were harvested by adding lysis buffer (Sigma-Aldrich) supplemented with 0.1% Triton-x-100 (Sigma-Aldrich).
Infection of cell lines treated with interferon. 5 x 10⁵ A549-ACE2 cells were seeded in 12-well plates. Twenty-four hours later, cells were infected with SARS-CoV-2 HK or Omicron at MOI 0.1, 1 or 5. As controls, we included untreated cells (NT) and cells infected with SARS-CoV-2 VLPs at MOI ~ 1. One-hour later, cells were washed, supplemented with fresh media, and treated with either DMSO or IFNα2a (1,000 U/mL; Sigma-Aldrich). Twenty-four hours post-infection, cells were harvested by adding lysis buffer (Sigma-Aldrich) supplemented with 0.1% Triton-x-100 (Sigma-Aldrich) and analyzed by western blot.

The liver was homogenized using the radioimmunoprecipitation assay lysis buffer (20–188; Millipore) containing a protease inhibitor (1183617001, Complete Mini EDTA‐free; Roche Life Science) and phosphatase inhibitor (04906837001, PhosSTOP phosphatase inhibitor cocktail; Roche Life Science). The homogenates were placed on ice for 60 min and centrifuged (4°C, 1500 g, 20 min). The total protein content of the samples was determined using a BCA protein assay kit (23227; Pierce). Proteins (10 μg of each sample) separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis were transferred to polyvinylidene difluoride membranes before being blocked for 60 min with 5% (w/v) bovine serum albumin in Tris‐buffered saline with 0.1% (v/v) Tween 20 (TBST). Membranes were incubated overnight at 4°C with the following primary antibodies: phospho‐glycogen synthase (GS) (p‐GS, Ser641, 3891; Cell Signaling Technology). After incubation, the membranes were washed in TBST, incubated for 1 h at room temperature with secondary antibodies (A102PT, American Qualex), and washed again in TBST. Chemiluminescent reagents (RPN 2232 and RPN 2109; GE Healthcare Japan) were used for blot detection. The blots were scanned and quantified using ChemiDoc XRS (170‐8071; Bio‐Rad) and Quantity One software (170‐9600; Bio‐Rad).

Whole-cell protein was isolated as described previously[42 (link)]. For tissue protein extraction, the tumor tissues were homogenized in ice-cold RIPA (radioimmunoprecipitation assay) lysis buffer (Millipore). The tissue lysates were incubated at 4°C for 1 hour with rotation, followed by clarification of tissue debris by centrifugation at 12,000 rpm for 10 minutes. The protein concentration of tumor extracts was determined using the BCA Protein Assay Kit (Pierce). Western blotting were performed as previously described[42 (link)]. Antibody binding was revealed using an HRP-conjugated anti-rabbit IgG or anti-mouse IgG (Sigma). Antibody complexes were detected using Immobilon Western Chemiluminescent HRP Substrate (Millopore) and exposure to X-Omat film (Kodak).