Goat anti rabbit hrp

The medium was removed and cells washed two times with ice-cold 1X PBS, then the COS-7 cells were collected in 1 mL of ice-cold 1X PBS by scraping, and the cell suspension was transferred to a 1.5-mL tube on ice. Then, the cell suspension in 1X PBS was sonicated on ice. The extracted cells were diluted 1:20 in 1X PBS and the protein concentration was measured with a Pierce^TM BCA Protein Assay kit from Thermo Fischer Scientific (#23225). Proteins were separated by SDS-PAGE using the Criterion system (BioRad, Hercules, CA, USA) and transferred to nitrocellulose membrane by wet Western blotting transfer in 50 mM Tris-base, 40 mM glycine, and 20% methanol. Blots were blocked by 5% skimmed milk in 0.05% PBST for 1 h, and washed with 0.05% PBST, then incubated with anti-GBA2 antibody (1:100), anti-FLAG antibody (1:1000), or anti-β-actin antibody (1:2000) as primary antibody overnight. After washing three times with PBST, goat anti-rabbit/HRP (Genscript) and rabbit anti-mouse/HRP (DAKO)-conjugated secondary were incubated with the blots to detect GBA2 (rabbit polyclonal antibodies) and Flag-tag primarily (rabbit monoclonal antibodies) and β-actin (mouse monoclonal antibodies) antibodies, respectively. After washing three times with PBST, the blots were developed with Luminata Forte Western HRP Substrate, according to the manufacturer’s instructions (Merck, Kenilworth, NJ, USA).

To detect the expression of ACE2 on the diltiazem-treated or CACNA1C-silenced cell surface, two 10-cm dishes of Vero-E6 cells were used to extract plasma membrane proteins by using the Minute Plasma Membrane Protein Isolation and Cell Fraction Kit (Invent Biotechnologies) following the manufacturer’s instructions. The total ACE2 in cells was extracted with RIPA buffer containing a protease inhibitor. Samples were incubated on ice for 30 min and centrifuged at 12,000 × g for 20 min at 4°C. Clarified cell lysate was diluted in denaturing SDS gel loading buffer and boiled for 15 min.
The samples were loaded onto a 4%–12% SDS-PAGE gel (Genscript) and separated by electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck-Millipore). The PVDF membrane was blocked with 5% skim milk and incubated with anti-Flag antibody (Genscript), anti-Myc antibody (Genscript), anti-ACE2 antibody (R&D system), anti-β-actin antibody (Zsbio), and anti-Zonula occludens protein 3 (ZO3) antibody (abcam). After being washed, the PVDF membrane was incubated with the following HRP-conjugated secondary antibodies: goat anti-rabbit HRP (Genscript), goat anti-mouse HRP (Genscript), and rabbit anti-goat HRP (Jackson ImmunoResearch). Signals were detected by using the enhanced chemiluminescence (ECL) reagent (Merck Millipore).