Ab51560

The procedures of Western blot were carried out as previously described [15 (link)]. Protein samples (20μg) were separated in an 8% DS-PAGE gel and transferred to a nitrocellulose membrane (#66485; PALL Life Sciences) via semi-dry transfer (350 mA, 90 min). Primary antibodies: Rabbit anti-TRPC3, 1:200, #ab 51560, Abcam; rabbit anti-Pan cadherin, 1:1000, ab6505, #ab16505, Abcam; mouse anti-GAPDH, 1:20,000, #2118S, Cell Signaling Technology. Densitometry was employed to quantify proteins of interest using the gel analyzer function of ImageJ (ver. 1.46a; National Institutes of Health, USA).

Isolated papillary muscles from interbout and summer ground squirrels were lysed in hypotonic buffer containing NaCl 20 mM, Tris-HCl 20 mM (pH 7.4) and 1% Triton X-100, supplemented with proteinase inhibitors and centrifuged at 2,500∙g for 5 minutes. Proteins were separated by denaturing 10% polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (0.45 μm; Santa-Cruz, sc-3724). Primary rabbit polyclonal antibodies (Abcam) against Trpc3 (ab51560) and Trpc5 (ab63151), mouse polyclonal antibodies against Trpc6 (ab63038) and Trpc7 (ab93618) and goat primary antibody against actin (Santa Cruz, sc-1616) were diluted at 1:1000 and used to probe immunoreactive proteins. Counterstain was performed with horseradish peroxidase (HRP)-conjugated anti-rabbit (Santa Cruz, sc-2004, 1:300 dilution), anti-mouse (Abcam, ab131368, 1:300 dilution) or anti-goat (Santa Cruz, sc-2020) secondary antibodies, respectively. HRP signals were detected using 3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate (Amresco, E733) and film-captured.

Fractions were quantified by D_C Protein Assay (Bio-Rad), and 30-40 μg was used for Western blot analyses by SDS-PAGE on 8% acrylamide gels. After blotting onto a PVDF membrane, the nuclear fraction was probed with anti-TFII-I mouse monoclonal antibody (610943; BD Biosciences) and normalized against nuclear loading control, nucleolin (ab22758; Abcam). The membrane fraction was probed with anti-TRPC3 rabbit polyclonal antibody (ab51560; Abcam) and normalized against membrane loading control, N-cadherin (610920; BD Biosciences). ImageJ (NIH, http://rsb.info.nih.gov/ij) was used to quantify the relative band densities.