Ab84115

Immunodetection of Ano1 was performed by Western blotting. Islets were isolated, as previously described, from two rats and lysed into 60 μl ice cold RIPA lysis buffer (50 mM Tris base, pH 7.4, 150 mM NaCl, 1 % NP-40, 0.25 % sodium desoxycholate, and supplemented with Roche protease inhibitor tablet). Protein concentration, measured by Bradford, was 4 μg/μl. After centrifugation (16,000 rcf, 15 min), samples were supplemented with Laemmli buffer/1 % dithiothreitol (Laemmli 4×: 250 mM Tris–HCl, pH 6.8, 40 % glycerol, 5 % SDS, bromophenol blue; sample: Laemmli buffer 4×, 3 v:1 v) and denatured by heating at 70 °C for 30 min. Proteins were separated on a 5 % acrylamide gel and transferred to a PVDF membrane. Procedure was as previously described [45 (link)]. After blocking, the membrane was successively incubated overnight at 4 °C with a rabbit polyclonal anti-ANO1 (ab84115, Abcam) 1:1000, rinsed, and incubated at room temperature for 2 h with HRP-conjugated anti-rabbit. Detection was performed by exposure to enhanced chemiluminescence (Amersham). Human thyroid extract was used as positive control. Molecular weight of rat Ano1 was estimated at 119 kDa, from Rattus norvegicus peptide sequence (NP_001101034.1, NCBI).

Six days old cultured normal myotubes were washed with 1x PBS, fixed in 4% paraformaldehyde both supplemented with 100 µM N-benzyl-p-toluene sulphonamide (BTS) and blocked by incubating in 5% goat or rabbit serum, as described in detail^{34 (link)}. Primary and secondary antibodies, diluted in PBS supplemented with 0.2% BSA and Triton X-100 were as follows: mouse monoclonal antibody 1A against DHPRα_1S (1:2,000, MA3-920, Affinity Bioreagents), rabbit polyclonal anti-ANO1 (1:50, ab84115, Abcam), goat polyclonal anti-ANO5 (1:500, sc-169628, Santa Cruz), rabbit anti-GFP (1:5,000, A11122, Invitrogen), secondary goat anti-mouse Alexa Fluor 594, goat anti-rabbit Alexa Fluor 488 (both 1:4,000, A11032 and A11034, respectively, Invitrogen), rabbit anti-goat Cy3 and rabbit anti-mouse FITC (1:500, C2821, and 1:2,000, F9137, respectively, Sigma).
Images were recorded with a cooled CCD camera (Diagnostic Instruments) and MetaVue image processing software (v 6.2, Universal Imaging, PA). Quantification of ANO1 surface membrane expression after siRNA knock-down was determined by measuring the average fluorescence intensity along the periphery of CFP positive myotubes, obtained from at least two different cultures using the MetaVue software.