Anti myc antibody

Clarified cell lysate was diluted in denaturing SDS gel loading buffer, and boiled for 15 min. After denaturing, the samples were loaded onto a 4 to 12% gel (GenScript, M41210C) for SDS-PAGE and separated by electrophoresis. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Merck-Millipore, ISEQ00010). The PVDF membrane was blocked with 5% skim milk in PBS containing 0.1% Tween 20, and then incubated with the following primary antibodies: anti-Flag antibody (GenScript, A00187), anti-Myc antibody (GenScript, A00172), anti-GST antibody (GenScript, A00097), or anti-His antibody (GenScript, A00174). The PVDF membrane was then washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (GenScript, A00160) and goat anti-rabbit antibody (GenScript, A00098). After three washes with PBST buffer, target protein bands were detected by using the enhanced chemiluminescence (ECL) reagent (Merck Millipore, WBLUR0500).

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).