Hrp conjugated goat anti mouse

Western blotting of ACE and ACE2 was carried out in the cell lysates of CD34+ cells as described before [16 (link)]. Cells were lysed in a radioimmuno precipitation assay (RIPA) buffer (Tris 10 mM pH 7.4, containing 140 mM NaCl, 1 mM EDTA, 1 mM NaF, 0.10% SDS, 0.50% sodium deoxycholate 0.1% NP-40, 1% Triton X-100) in the presence of protease inhibitors (Thermo Fisher). Protein concentration in cell lysates and cell supernatants was determined using bicinchoninic acid with bovine serum albumin as a standard (Thermo Fisher). Equal amounts of protein (30 μg) were loaded and separated by SDS-PAGE using SurePage 10% pre-casted gels (Genescript). Proteins were electro-blotted onto nitrocellulose membranes (Bio-Rad). The blots were blocked using 5% (w/v) milk in Tris-buffered saline containing 0.5% (v/v) Tween-20. The membranes were then incubated with different antibodies. The ACE2 and ACE antibodies that were used are ab87436 and ab28311, respectively, with a β-actin antibody (mab8929; R&D Systems). HRP-conjugated goat anti-mouse (Biolegend) or donkey anti-rabbit (406–401; Biolegend) secondary antibodies were used at 1:20,000 dilution. Enhanced chemiluminescence reagent (ECL, K15045-D50; Advansta) was used to visualize bands and developed on X-ray films (Phoenix research products). ImageJ (NIH) was used for quantification of band intensities.