Mouse anti muc5ac

Lung tissue (200 mg) was homogenized in RIPA lysis buffer (100 ml PBS 1x pH 7.2, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, supplemented with protease inhibitors). Total protein was quantified using Bradford assay (Bio-Rad, Hercules, CA). Samples were subjected to SDS-PAGE electrophoresis, using a 15% acrylamide gel for CC10 and 10% acrylamide for Notch1. Seventy five μg of protein were loaded for CC10 and 50 μg for Notch1. They were transferred to PVDF membranes and blocked with 5% nonfat milk. The following primary antibodies were used: mouse anti-Muc5ac (1:2000, sc-21701, Santa Cruz Biotechnology), goat anti-Muc5b (1:2000, sc-135508, Santa Cruz Biotechnology), rabbit anti-CC10 (1:4000, ab40873, Abcam, UK), goat anti-Notch1 (1:2000, sc-6015, Santa Cruz Biotechnology), and Anti-Actin I-19 (1:2000, sc-1616, Santa Cruz Biotechnology, USA). Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) were used. Brain and stomach were used as positive controls. Water without antibodies was used as negative control. Proteins were detected by Pierce, ECL Western Blotting Substrate (Pierce Biotechnology, Rockford, IL). Images were film-captured. Density of band was measured using Fiji software (ImageJ 2.0.0-rc-54/1.51h) and protein amounts were normalized with the actin signal.

Primary antibodies used were rabbit monoclonal anti-SARS-CoV-2 nucleocapsid (NP) (clone 001) (catalog no. 40143-R001; SinoBiological US, Wayne, PA) at a dilution of 1:25, mouse monoclonal anti-β-tubulin IV antibody (clone ONS.1A6) (catalog no. T7941; MilliporeSigma, St. Louis, MO) at 1:100, mouse anti-ZO-1 (clone 1/ZO-1) (catalog no. 610966; BD Bioscience, San Jose, CA) at 1:100, and rabbit anti-Ki67 (clone SP6) (ab1666; Abcam, Cambridge, MA) at 1:50. Mouse anti-MUC5AC (Santa Cruz Biotechnology, catalog no. sc-33667; 1:10), mouse anti-cytokeratin k5 (ThermoFisher Invitrogen, catalog no. MA5-12596; 1:50), rat anti-SCGB1A1 (ThermoFisher Invitrogen, catalog no. MAB4218; 1:50), and goat anti-ACE2 (Novus Biologicals, catalog no. AF933, 1:10) were used to mark epithelial cell types.