Las 4000 mini apparatus

The virus-infected MDCK or VeroE6/TMPRSS2 cells in 6-well plates were washed with PBS and lysed in 500 μl of 1× TNT buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol). Lysates were centrifuged at 20,630 × g for 10 min at 4 °C. The supernatant was mixed with 4× lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, NP0007) and 10× sample reducing agent (Invitrogen, NP0009). Samples were boiled for 5 min and fractionated by NuPAGE 10% Bis-Tris Protein Gels (Invitrogen, NP0316BOX) and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, 170-4156). The membranes were incubated with mouse anti-influenza A virus NS1 (Santa Cruz, sc-130568; 1:1000), mouse anti-influenza A virus M2 (Santa Cruz, sc-32238; 1:1000), mouse anti-α tubulin (Santa Cruz, sc-32293; 1:2000), or rabbit anti-SARS-CoV-2 nucleocapsid (Cell Signaling, 33336; 1:1000) antibody, followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG (Jackson Immuno Research Laboratories, 115-035-003; 1:10,000) or anti-rabbit IgG (Invitrogen, G-21234; 1:10,000). The PVDF membranes were then treated with Chemi-Lumi One Super (Nacalai Tesque, 02230-30) to elicit chemiluminescent signals, which were detected and visualized using an LAS-4000 Mini apparatus (GE Healthcare).

Colon tissues were lysis harvested on ice by washing twice with cold PBS, scraping, and resuspending. The protein concentration of colon lysate was determined using the BCA assay with bovine serum albumin. Protein samples prepared by mixing SDS sample buffer were run on NuPAGE 4–12% gradient Bis-Tris and electrotransferred to a polyvinylidene difluoride membrane (Thermo Fisher Scientific) as in a previous study.64 Blots were blocked with 5% (w/v) skim milk in Tris-buffered saline solution containing 0.1% Tween 20 (Pierce, Appleton, WI, USA) and incubated overnight at 4 °C with antibodies against Reg3β (1:1000, R&D Systems) and β-actin (Sigma). Blots were additionally incubated with secondary antibodies conjugated with horseradish peroxidase for 1 hour at room temperature and visualized with ELC Prime detection reagent and the LAS 4000 Mini apparatus (GE Healthcare, Piscataway, NJ, USA).