Ab53212

The thalamus and kidney from BALB/c mice, as well as ANO2-EGFP-transfected and untransfected NIH/3T3 cells, were lysed in RIPA buffer (Tris-base, 6.5 mM; NaCl, 15 mM; EDTA, 1 mM; NP-40, 1%; and Na-deoxycholate, 0.25%) with 1 mM phenylmethyl sulphonyl fluoride, Na₃VO₄, NaF and protease cocktail. Lysates were centrifuged at 13,000 r.p.m. for 10 min at 4 °C, and the supernatants were isolated for SDS–PAGE. From each sample, 50 μg of protein was resolved on 8% polyacrylamide gels and subsequently electroblotted to polyvinylidene fluoride (Bio-Rad, USA) membranes. Blots were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-linked secondary antibodies. Signals were visualized using ECL Plus reagent (Thermo Scientific, USA). The following antibodies were used: rabbit anti-ANO1 (ab53212, 1:500, Abcam, UK), rabbit anti-ANO2 (1:500 (ref. 35 (link))), mouse anti-β-actin (sc-47778, 1:2,000, Santa Cruz, USA), goat anti-rabbit-HRP-conjugated (31460, 1:5,000, Pierce, USA) and goat anti-mouse-HRP-conjugated (31430, 1:5,000, Pierce).

The thoracic aortas were isolated and embedded in optimal cutting temperature compound (Sakura, Japan) for sectioning at an 8-um thickness. Frozen slides were incubated overnight at 4ºC with antibodies against LC3B (NB100-2220; 1:100; Novus Biologicals, CO, USA) and alpha-smooth muscle actin (α-SMA) (BM0002; 1:100; Boster Biological Technology) and then treated with FITC-labeled anti-rabbit IgG (31635; 1:100) and Cy3-labeled anti-mouse IgG (A10521; 1:100) secondary antibodies (Invitrogen, CA, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). MASMCs were infected with the mRFP-GFP-LC3 adenovirus for 48 h. The puncta in thoracic aortas and MASMCs were viewed under a confocal microscope (Zeiss LSM800, Carl Zeiss, Munich, Germany) with z-stacks and 63× objective lens. The number of puncta was analyzed using the ImageJ program. For endogenous colocalization studies, MASMCs were incubated with TMEM16A (ab53212; 1:50), VPS34 (ab124905; 1:50) (Abcam), p62 (sc-48402; 1:100), Beclin-1 (sc-48341; 1:1000) (Santa Cruz Biotechnology), and Bcl-2 (BM4985; 1:100) (Boster Biological Technology) antibodies, and processed for colocalization observation with a confocal microscope.

Proteins from the cell lysates were prepared from cell lines as previously described [16 (link)]. Equal amounts of protein were denatured and separated by SDS-PAGE, transferred onto PVDF membranes, and incubated with polyclonal antibodies to TMEM16A (Abcam, ab53212; 1:100) and mouse β-actin antibody (Cell Signalling; 1:500). The peak intensity of each band was visualized using an Enhanced Chemiluminescence kit (Amersham, Little Chalfont, UK) on X-ray film (Millipore Corporation, Billerica, USA).

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and dissolved in lysis buffer (150 mM NaCl, 20 mM Tris, 5 mM EDTA, pH 7.5, 1% Triton X-100 and supplemented with 1 mM PMSF). After protein quantification, samples containing 100 µg proteins were denatured and electrophoresed using 10% SDS-PAGE and then transferred onto PVDF membranes. After blocking with 5% (w/v) nonfat milk and washing with Tris-buffered saline–Tween solution (TBST), the membranes were incubated overnight at 4°C with primary anti-TMEM16A polyclonal antibody (Abcam, ab53212; 1∶100) and mouse anti-β-actin antibody (Cell Signaling; 1∶500), respectively. All other primary antibodies were purchased from Cell Signaling Technology. After washing, the blots were incubated with secondary antibody conjugated to horseradish peroxidase (Sigma; 1∶5,000) for 1 h at room temperature and visualized using an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK) on X-ray film (Millipore Corporation, Billerica, USA).

The SG tissue were harvested at CT0, 6, 12, 18, 24, 30, 36, 42 and 48, and homogenized in ice cold radioimmunoprecipitation assay (RIPA) lysis buffer (188–02453, Fujifilm-wako Corp., Osaka, Japan). Protein samples concentration were calculated using the DC protein assay kit (Bio-Rad, Richmond, CA) based on the Lowry method. For each sample, 10 μg protein was electrophoresed on 10% SDS-PAGE gel, transferred to Immobilon-P Transfer Membrane (PVDF, Millipore, Burlington, Massachusetts, USA) membrane and analyzed using the Mini Trans-Blot® Transfer Cell (#1703930JA, Bio-Rad, Richmond, CA). PVDF membrane were blocked with 5% skimmed milk for 1h, and probed overnight at 4 °C with anti-ANO1 (1:500, ab53212; Abcam, Cambridge, UK), anti-Aquaporin 5 (1:5000, ab78486; Abcam, Cambridge, UK) and anti-β ACTIN (1:10000, GTX110564; GeneTex, Alton Pkwy, US). Horse-radish peroxidase (HRP)-conjugated polyclonal goat anti-rabbit immunoglobulins (1:1000, P0448; Dako, California, US) was used for 1 h at room temperature. Protein bands were visualized with the ECL chemiluminescence WB Detection Reagents (GE Health Care, Little Chalfont, UK), and documented using the Image Quant LAS-4000 (GE Health Care). Quantification of bands were performed by using Image Quant TL 7.0 software (GE Health Care).

Cell lysates were prepared using Triton X-100 lysis buffer (150 mM NaCl, 20 mM Tris, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM EDTA) containing cocktail protease inhibitor (Roche Applied Science). Protein samples were denatured at 95°C for 5 minutes, separated by SDS-PAGE and then transferred to PVDF membranes (Millipore). After blocking with 5% milk for 1 hour, the membranes were incubated with primary antibodies against ANO1 (1:1000; ab53212, Abcam), Caspase-7 (1:1000; 12742, Cell Signaling Technology), CDK2 (1:500; sc-6248, Santa Cruz), Cyclin D1 (1:1000; 2978, Cell Signaling Technology) or actin (1:2000; TA-09, Zhongshan golden bridge) overnight at 4°C. After extensive washing in TBS buffer, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. The signal was detected using an ECL Western blotting detection system (Millipore).