Acc 010

After infection, cells were fixed for 15 min with 3.7% formaldehyde, permeabilized with Triton X-100 (0.25% in 5% FBS/PBS) for 10 minutes and blocked with 5% FBS/PBS for 1 hour at room temperature. Primary rabbit antibodies: anti–IRF-7 antibody (1:200, ab62505, Abcam), anti-PapG pre-absorbed serum (1:1,000), anti-TWIK-1 (1:50 sc-28630, Santa Cruz biotechnologies), anti-TRAAK (1:50, sc-50413, Santa Cruz biotechnologies), anti-KCNJ2 (1:100, 3305–1, Epitomics), anti-KCNJ11 (1:100 APC-202, Alomone Labs), anti-TRPC1 (1:100, ACC-010, Alomone Labs), anti-TRPV6 (1:250 orb158655, Biorbyt) and secondary goat anti-rabbit Alexa Fluor 488–conjugated antibody (1:200, A-11034, Thermo Fisher Scientific) were used. Nuclei were stained with DRAQ-5 (ab108410, Abcam). Slides were mounted using Fluoromount and examined in a LSM 510 META laser-scanning confocal microscope (Carl Zeiss). Fluorescence was quantified using the ImageJ software.

After infection, cells were lysed with NP-40 lysis buffer, supplemented with protease and phosphatase inhibitors (both from Roche Diagnostics). Total cellular proteins were run on SDS–polyacrylamide gel electrophoresis (4 to 12% bis-tris gels; Invitrogen), blotted onto poly-vinylidene difluoride membranes (GE Healthcare), blocked with 5% non-fat dry milk (NFDM), and incubated with rabbit anti–IRF-7 (1:300, ab62505, Abcam, Cambridge, United Kingdom) rabbit anti-TRPC1 (1:500, #ACC-010, Alomone Labs) and rabbit anti-TRPV6 (1:500, #orb158655, Biorbyt) antibodies. The blots were washed with PBS Tween 0.1% (PBST) and incubated with HRP-linked secondary antibodies in 5% NFDM (1:4,000, goat anti-rabbit- horseradish peroxidase (HRP), #7074, Cell Signaling). The anti-β-actin (1:4,000 in 5% NFDM, #A1978, Sigma-Aldrich) followed by rabbit anti-mouse Immunoglobulins HRP-linked (1:4,000 in 5% NFDM, P0260, Dako) was used as loading control. The blots were washed with PBST and developed with ECL Plus detection reagent (GE Healthcare). Blots were imaged using the Bio-Rad ChemiDoc System (Bio-Rad) and quantification of densitometry of bands was done using the ImageJ software (NIH).

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.