Image acquisition system

Total proteins were extracted from cells using RIPA buffer (10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors cocktail from Sigma). The lysates were centrifuged 15,000 × g at 4°C for 20 min and mixed with a La $\ddot{\mathrm{e}}$ mmli buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 5% β-mercaptoethanol, 20% glycerol, and 0.01% bromophenol blue) and boiled for 5 min at 95°C. Total protein samples were separated using 10% SDS-PAGE and transferred to a PVDF membrane with Power Blotter XL (Invitrogen, Massachusetts, United States). Membranes were incubated with anti-TRPV6 [1:500, (Haustrate et al., 2023a (link))] and anti-β-actin (Sigma-Aldrich, 1:1000, A5441). Immunocomplexes were visualized through the enhanced chemiluminescence method (Pierce Biotechnology Inc., Massachusetts, United States). Densitometric analysis was performed using a Bio-Rad image acquisition system (Bio-Rad Laboratories, CA, United States).

SW480 and HCT116 cells were seeded in 6-well plates until they grew with adherence, the various conditioned media was replaced as described previously and incubated for 24 hours, and cell extracts were prepared in ice-cold lysis buffer containing protease inhibitor. The cell proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride (PVEF) membranes (Millipore). Membranes were further incubated sequentially with specific antibodies including anti-E-cadherin (1:1000, Pro780, Cell Signaling Technology), anti-N-cadherin (1:1000, EPR1791–4, Epitomics), anti-Vimentin (1:1000, EPR3776, Epitomics), anti-MIF (1:1000, Santa Cruz Biotechnology), anti-p-Cofilin (1:1000, 77G2, Cell Signaling Technology), and anti-F-actin (5 μg/ml, 4E3.adl, Abcam). After primary antibodies were incubated, the blots were subsequently incubated with appropriate secondary antibodies. Protein bands were visualized with ECL reagent (Thermo Scientific Inc.) and a Bio-Rad image acquisition system (Bio-Rad Laboratories). The protein bands was quantified using densitometric scanning software, and relative protein abundance was determined by normalization with tubulin or GAPDH.

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.

Total protein was extracted from the SNpc tissues of rats and quantitated using a bicinchoninic acid (BCA) assay. Proteins were separated with SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked by 5% nonfat dry milk for 1 h and incubated with primary antibody (anti-p38 MAPK, diluted 1:1,000) over night. Then, the respective secondary antibody was incubated for 1 h at room temperature. Densitometric analysis was performed using a Bio-Rad image acquisition system (Bio-Rad Laboratories). β-Actin was used as an internal reference to detect the relative expression of p38 MAPK protein.

Semiconfluent cells were treated with an ice-cold lysis buffer containing the following: 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 10 mM MgCl₂, 1 mM PMSF, 1% Nonidet P-40, and protease inhibitor cocktail from Sigma. The lysates were centrifuged 15,000× g at 4 °C for 20 min, mixed with a sample buffer containing 125 mM Tris-HCl pH 6.8, 4% SDS, 5% β-mercaptoethanol, 20% glycerol, and 0.01% bromophenol blue, and boiled for 5 min at 95 °C. Total protein samples were subjected to 8% SDS-PAGE and transferred to a nitrocellulose membrane by semi-dry Western blotting (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked in a 5% milk containing TNT buffer (Tris-HCl, pH 7.5, 140 mM NaCl, and 0.05% Tween 20) overnight then probed using specific rabbit polyclonal anti-TRPV6 antibodies (all at 1/500 dilution from the initial concentration of 0.5 µg/µL) and mouse monoclonal anti-β-actin (Lab Vision Co., Fremont, CA, USA, 1/1000) antibodies. Goat polyclonal anti-rabbit and anti-mouse peroxidase-conjugated secondary antibodies (Chemicon International; Temecula, CA, USA, 1/200) were used. The bands on the membrane were visualized using enhanced chemiluminescence method (Pierce Biotechnologies Inc., Escondido, CA, USA). Densitometric analysis was performed using a Bio-Rad image acquisition system (Bio-Rad Laboratories).