Minute plasma membrane protein isolation and cell fraction kit

HEK293 cells were transfected with plasmids by using TransIT-293 transfection reagent according to the manufacturer’s instructions. Vero-E6 cells were transfected with plasmids by using ExFect transfection reagent (Vazyme, T101-AA) according to the manufacturer’s instructions. At 48 h posttransfection, cells were lysed with the gentle extraction buffer, NP-40 lysis buffer, or the rigorous extraction buffer, RIPA lysis buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) for 1 h at 4°C. Supernatant was collected and mixed with 40 μL of protein G agarose for 4 h at 4°C to remove nonspecific binding proteins in the supernatant. After being washed, the supernatant was mixed with anti-Flag antibody-conjugated agarose beads for 6 h at 4°C. The beads were isolated by centrifugation, washed five times with lysis buffer, and used for SDS-PAGE and Western blotting.
To detect the interaction between mGluR2-Flag and endogenous TfR1, HEK293 cells expressing mGluR2-Flag protein from two 15 cm dishes were used to extract cell plasma membrane by using the Minute Plasma Membrane Protein Isolation and Cell Fraction Kit (Invent Biotechnologies, SM-005). The yields were then used for the co-immunoprecipitation assay, SDS-PAGE, and Western blotting.

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).