Pa1 901

Protein-protein interactions were visualized using Duolink In Situ detection kit (DUO92002/4/7; Sigma-Aldrich) by following the manufacturer’s protocol. Briefly, cells were cultured on 12 mm glass coverslips in 24 well plates. At the end of the experiment, cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized (0.2% Triton X, 0.1 M glycine in PBS). Next, the cells were incubated with blocking solution, followed by overnight incubation with primary antibodies. The cells were then washed, probed with the secondary antibodies conjugated with oligonucleotide, ligated, and amplified. Preparations are then mounted using Duolink II mounting medium containing DAPI (DUO82040; Sigma-Aldrich), and the fluorescence signals were analyzed by ImageXpress Micro confocal and MetaXpress software (Molecular devices). Primary antibodies used in in situ PLA experiments were anti-rabbit VDAC1 (Abcam, ab15895; 1:100 dilution), anti-mouse monoclonal IP3R1 (Santa Cruz; sc-271197; Clone E8; 1:50 dilution), anti-mouse monoclonal GRP75 (Santa Cruz, sc133137; Clone D9; 1:50 dilution), anti-rabbit IP3R1 (Invitrogen, PA1-901; 1:100 dilution), anti-rabbit FLAG (Cell signaling, #2368; 1:100 dilution) and anti-mouse HA (Santa Cruz, sc7392; Clone F-7; 1:50 dilution).