Sc 32949

MCF-7 cells were lysed in RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCl pH 7.4, 2 mM EDTA, 0.5 mM sodium orthovanadate, and P8340 inhibitor cocktail (Sigma-Aldrich)). 1 mg of MCF-7 protein lysates were precleared for 30 min with proteins A and G sepharose magnetic beads and then incubated over night with 3 μg of anti-KCa3.1 antibody (sc-32949, Santa Cruz, Santa Cruz, CA). The antigen-antibody complexes were precipitated with proteins A and G sepharose magnetic beads (Millipore, PureProteome™ Magnetic Beads) for one hour. After denaturation, proteins were separated by denaturing SDS–PAGE and transferred onto nitrocellulose membranes. KCa3.1 was detected using anti-KCa3.1 antibody (sc-32949, Santa Cruz, at 1:500) and anti-rabbit secondary antibody (TrueBlot Anti-Rabbit IgG HRP 18-8816, eBioscience, Paris, France, at 1:1000). Anti-TRPC1 anti-body (ACC-010, Alomone, Jeruzalem, Israel, at 1:200) was used for TRPC1 detection. Bands were detected using an enhanced chemiluminescence kit (GE Healthcare, Saclay, France) and quantified using the densitometric analysis option in the Bio-Rad image acquisition system (Bio-Rad Laboratories, Marnes-la-Coquette, France). For lipid rafts disruption induced by surface cholesterol depletion, MCF-7 cells were treated with 5 mg/mL of Methyl-β-cyclodextrin (Sigma-Aldrich) for 24 h in complete culture medium.