Mouse monoclonal anti alpha tubulin antibody

The airway epithelium cultured on 6.5-mm Transwell membranes was washed with PBS, scraped out of all the cells, and pelleted by centrifugation at 10,000 rpm for 5 minutes. The cell pellet was incubated with 1x LDS loading buffer (Thermo Fisher Scientific) with proteinase inhibitor (Roche), transferred into a QIAshredder microcentrifuge tube, and centrifuged for 3 minutes at 15,000 rpm in a tabletop centrifuge. The elusion from the QIAshredder was collected and stored in a freezer. The protein concentration was measured using a BCA protein assay kit (Thermo Fisher Scientific). For the detection of ACE2, total protein (30 μg) was separated on 4–12% Bis-tris SDS polyacrylamide gels (reducing) and then subjected to dry blot transfer onto PVDF membranes according to the manufacturer’s instructions (Life Technologies). The PVDF membrane was imaged using an Odyssey CLX system (Li-Cor Biosciences). ACE2 was detected by Western blotting using anti-ACE2 goat polyclonal antibody (R&D Systems) and corresponding donkey anti-goat IRDye 800 secondary antibodies (Li-Cor Biosciences). For the loading control, alpha-tubulin was detected by anti-alpha-tubulin mouse monoclonal antibody (Sigma-Aldrich) and the corresponding goat anti-mouse IRDye 680 secondary antibody (Li-Cor Biosciences). Image Studio 5.2 (LI-COR Biosciences) was used to quantify the protein signal.

Cells were homogenized in lysis buffer (50 mM TrisHCl, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% NP-40, 1 mM PMSF and 1 μg/mL pepstatin A). Protein concentration was measured with the BCA (bicinchoninic acid) assay (Thermo Fisher, Waltham, MA). Equal amounts of protein were loaded in 15% SDS gel, separated by electrophoresis and transferred to PVDF membranes (polyvinylidene difluoride, Millipore, Darmstadt, Germany). The membranes were blocked with 5% TBS/0.5% v/v Tween-20 skim milk and incubated with anti-caspase3 (1:1000, Cell Signaling Technology, Danvers, MA), anti-BcLxL (1:250, Santa Cruz), anti-Bax (1:100, BD Pharmingen, San Jose, CA), anti-LC3 (1:1000, Novus Bio, Centennial, CO) or anti-heme oxygenase (HO-1, 1:1000, Enzo Life Technologies, Farmigdale, NY) antibodies dissolved in 5% milk PBS/Tween for 1h at room temperature. They were then washed with TBS/Tween and incubated with the secondary antibodies against rabbit IgG (1:5000) or mouse IgG (1:5000). After washing with PBS/Tween, blots were developed with the chemiluminescence method (ECL; Thermo Fisher) and probed with mouse monoclonal anti–alpha-tubulin antibody (1:10000, Sigma-Aldrich). Levels of expression were corrected for minor differences in loading.