Westsave gold detection reagents

Tissues were lysed in RIPA buffer (R4100-010, Gendepot, Barker, USA) supplemented with protease inhibitor cocktail (P3300-001, Gendepot) on ice and homogenized with stainless steel beads (Qiagen, USA) in TissueLyser II (Qiagen). The lysates were added to sample loading dye NuPAGE^™ LDS sample buffer, heated to 95°C for 10 min and were separated on precast polyacrylamide gels (Bolt 4–12% Bis-Tris Plus, Thermo Fisher Scientific Inc., USA). After separation, proteins were transferred to a nitrocellulose membrane. After blocking the nonspecific site with 3% bovine serum albumin (BSA), the membrane was then incubated with anti-hACE2 antibody (1:2000, ab108209, Abcam) in 3% BSA at 4°C for overnight. The membrane was further incubated for 1 h with a peroxidase-conjugated secondary antibody (1:5000, Santa Cruz, USA) at room temperature. Immunoactivity proteins were detected with WESTSAVE Gold detection reagents (LF-QC0103, AbFrontier, Republic of Korea).

Standard sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and Western blotting were used to analyze the expression of various proteins. Cells were lysed in the lysis buffer (Cell Signaling Technology, Cat#. 9803) containing protease inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland). The quantitative protein concentration was determined by BCA Protein Assay Kit (Thermo Fisher Scientific, Cat#. 23225) and equal amounts of protein were loaded on 8–12% SDS/PAGE. Proteins were transferred to polyvinylidene difluoride membrane (Merck Millipore, Burlington, MA, U.S.A.) and subjected to immunoblotting using various antibodies overnight at 4°C, followed by further incubation with the secondary antibody (AbFrontier, Seoul, Korea, Cat#. LF-SA8001 and LF-SA8002) at room temperature for 1 h. Visualization of protein bands was detected with Westsave Gold detection reagents (AbFrontier, Cat#. LF-QC0103).

Standard sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting were used to analyze the expression of various proteins according to the procedure described previously [40 (link)]. Cells were homogenized, washed with ice-cold PBS, and lysed by cell lysis buffer (Cell Signaling Technology, Cat#. 9803) supplemented with complete EDTA-free protease and phosphatase inhibitors cocktails (Roche, CA, U.S.A.). The quantitative protein concentration was determined by BCA Protein Assay Kit (Thermo Scientific, MA, U.S.A.) and equal amounts of protein were loaded on 8–12% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membrane (Merck Millipore, MA, U.S.A.) and subjected to immunoblotting using various antibodies overnight at 4°C followed by further incubation with the secondary antibody (AbFrontier, South Korea, Cat#. LF-SA8001 and LF-SA8002) at room temperature for 1 h. Visualization of protein bands was detected with Westsave Gold detection reagents (AbFrontier, Cat#. LF-QC0103).