Sc 28614

The Proximity Ligation Assay (PLA) was performed as previously described (Paillard et al., 2013 (link); Pauly et al., 2017 (link)). Cells were fixed with paraformaldehyde 4% and permeabilized at RT with 0.1% Triton-X100. After washing, they were incubated with blocking buffer for 30 min at 37°C. The blocking solution was removed before incubation of primary antibodies (anti-VDAC Abcam, ab1734, 1:200, and anti-IP3R1, Santa Cruz, sc-28614, 1:200, or anti-GRP75, Santa Cruz, sc-13967) overnight at 4°C. The cells were washed two times using PBS with 0.01% Tween. The two PLA probes 1:5 were prepared in antibody diluent 20 min before incubation for 1 h at 37°C. Next, cells were incubated with mix containing 5× ligation stock (diluted 1:5 in water) and 1× ligation solution (diluted 1:40) for 30 min at 37°C. Next, the cells were incubated with mix containing 5× amplification stock (diluted 1:5 in water) and polymerase (diluted 1:80) for 100 min at 37°C. Finally, the cells were washed with Dapi (diluted 1:500) in wash buffer B 1× and mounted using Dako fluorescent mounting medium (S3023) and analyzed using a fluorescence microscope (excitation: 594 nm, emission: 624 nm, magnification: 40×).

For Western-blotting, cell lysates were obtained by treating the cell monolayer with RIPA buffer complemented with protease and phosphatase inhibitors. Protein lysates were then cleared by centrifugation (17’000g for 20min). Total protein concentration was determined using bicinchoninic acid protein assay (Interchim, UP40840) and 25μg of denatured and reduced proteins of each sample was loaded on a 6% SDS-PAGE. For the IP3R1 immunoblotting (1/500, Abcam, ab5804) (S1B Fig), a 10% SDS-PAGE gel was used and the protein transfer was performed at low intensity overnight in a cold room. For the IP3R3 immunoblotting (1/1000, BD Biosciences, 610312) (S1C Fig), a 10% SDS-PAGE gel was used and the protein transfer was performed at low intensity overnight in a cold room.
After SDS-PAGE migration and electroblotting on polyvinylidene fluoride, the membranes were blocked with 5% non-fat milk and then incubated with the specific primary antibodies [rabbit anti-IP3R1 (Santa-Cruz, sc-28614; 1/500) and rabbit anti-TUBULIN (Santa-Cruz, sc-5286; 1/500)] (S1A Fig).
Blots were incubated with horseradish peroxidase (HRP)-coupled sheep anti-mouse IgG (GE Healthcare, NA931VS; 1/10000) and (HRP)-coupled goat anti-rabbit IgG (GE Healthcare, NA934VS; 1/10000), and developed with Clarity Western ECL Substrate (BioRad, 1705060). The band intensity was determined using Image Lab software (Bio-Rad).