Amersham ecl select

MCs treated as described above were washed with ice‐cold PBS and lysed for 5 min on ice with lysis buffer (10 mmol/L Tris at pH 8.0, 150 mmol/L NaCl, 0.2 mmol/L sodium orthovanadate, 0.1% Triton X‐100, 0.5 mmol/L IGEPAL, and 1 protease inhibitor tablet, EDTA‐free from Thermo Fisher Scientific, Waltham, MA). The lysates were then cleared by centrifugation at 21,300g for 30 min at 4°C, and total protein was quantified with a Pierce^™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (15–20 μg) were separated on 10% polyacrylamide gels and electrotransferred to nitrocellulose membranes (Amersham Hybond ECL; GE Healthcare, Little Chalfont, UK) that were subsequently blocked for 1 h at room temperature in Tris‐buffered saline‐Tween (20 mM Tris‐base at pH 7.6, 150 mmol/L NaCl, and 0.1% Tween 20) containing 5% BSA. The membranes were then incubated with an anti‐ACE (H‐170, 1:500) or anti‐ACE2 (H‐175, 1:500) antibody (Santa Cruz Biotechnology, Dallas, TX) in blocking solution at 4°C overnight, before being washed and incubated with a diluted conjugated secondary antibody at room temperature for 1 h. Antibody binding was visualized using an enhanced chemiluminescence kit (Amersham ECL Select^™; GE Healthcare), and chemiluminescent signals were quantified using ImageJ software.

Monolayer cells were solubilized with 20 mM Tris buffer (pH 8.0) containing 1% SDS, 1 mM phenylmethylsulfonyl fluoride, 125 mU/mL Benzonase Nuclease (Merck Millipore Co., Tokyo, Japan) (25 µL/cm² culture surface area). The lysate (10 µL/lane) was resolved by SDS-PAGE under reducing conditions using 6% polyacrylamide gels or 4–15% precast gels (Bio-Rad Laboratories Inc., Hercules, CA, USA). Then it was transferred to a polyvinylidene difluoride membrane (Immobilon-P; Merck Millipore Co.) by semi-dry blotting in a buffer containing 25 mM Tris, 192 mM glycine, 0.1% SDS, and 20% methanol. The membrane was blocked with 5% non-fat milk in 20 mM Tris-buffered saline (pH 7.2) containing 0.1% Tween 20 and reacted with 1 µg/mL of mAb SKM9-2 or anti-FLAG (Anti-DYKDDDDK tag mAb; 1E6, Wako Pure Chemical Industries, Osaka, Japan). After washing, it was treated with horseradish peroxidase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA, USA), and developed with Amersham ECL select (GE Healthcare, Buckinghamshire, UK). Although glycan in non-fat milk often interferes the glycan-dependent binding of an antibody, the SKM9-2 binding was not affected by the blocking using non-fat milk, in comparison with 5% bovine serum albumin (Supplementary Fig. S6).

Protein expression and purification was confirmed by SDS–polyacrylamide gel electrophoresis (PAGE) with 12.5% or 17% gel, followed by staining with Coomassie brilliant blue staining solution, EzStain Aqua (ATTO, Tokyo, Japan). For Western blotting, proteins after SDS-PAGE were transferred on PVDF membrane, followed by blocking with Block Ace (KAC Co., Ltd., Kyoto, Japan). Anti-flavi group antigen antibody (D1-4G2-4-15, Merck, Darmstadt, Germany), anti-DYKDDDDK tag antibody (Wako, Osaka, Japan) and anti-GAPDH antibody (Santa Cruz Biotechnology, Dallas, USA) were used as the primary antibodies. Membranes were then washed with Tris buffered saline containing 0.1% Tween 20 and treated with anti-mouse IgG HRP linked antibody (Cell Signaling Technology, Danvers, USA). Protein bands were stained with Amersham ECL Select (GE healthcare, Chicago, USA). Pictures of stained gels and membranes were obtained with Chemidoc touch (Bio-Rad, Hercules, USA).