Tx 160

In total, 1.25 mL of pseudoviruses was pelleted at 4 °C for 2 h at 15,000 rpm using Tomy TX-160 ultracentrifuge. Pseudovirus pellet was resuspended in 1× Laemmli loading buffer and heated at 70 °C for 10 min. Samples were resolved by 4–20% SDS PAGE and transferred onto nitrocellulose membranes. Membranes were probed for SARS-CoV-2 S1 using rabbit polyclonal antibodies against the RBD domain (Sino Biological, Wayne, PA, USA).

IB was performed as described before [32 (link)]. Briefly, top-most portion of left lungs were excised and placed in ice-cold HBSS. Equal weight of tissue (approximately 100 mg of tissue from each mouse) were sonicated in RIPA (25 mM Tris, 150 mM sodium chloride [Sigma], 1% NP-40 [Sigma], 1% sodium deoxycholate [Sigma], and 0.1% SDS [Sigma], pH 7.6; 200 μl per sample) supplemented with protease inhibitor and phosphatase inhibitor cocktail (Roche). After sonication, tissues were lysed on ice for 30 mins, collected by centrifugation at 150,000 rpm at 4°C for 20 mins using a Tomy TX-160 high speed refrigerated micro centrifuge. The resulting supernatant (tissue lysate) was collected for IB analysis. Beta actin levels were used to determine comparable protein quantities in all samples for IB. Proteins were transferred to PVDF membranes (Bio-Rad), treated with chemiluminescence reagent (Clarity, Bio-Rad) for signal development and imaged on Kwik Quant Gel Imaging System (Kindle Biosciences Inc). Antibodies used were rabbit anti-cleaved Casp8 (8592, Cell Signaling Technology), rabbit anti-cleaved Casp3 (9661, Cell Signaling Technology) and mouse anti-β-actin (A2228, Sigma).