Sc 13967

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×).

Western blotting analyses were performed with mouse anti-GAPDH (1:2,000, MAB374, Millipore), mouse anti-β actin (1:2,000, AC-74 Sigma), rabbit anti-TOM20 (1:1,000, sc-11415, Santa Cruz), rabbit anti-Tubulin (1:1,000, RB-9281-P0, Thermo), mouse anti-Opa1 (1:500, 612606, BD Biosciences), rabbit anti-GRP75 (1:1,000, sc-13967, Santa Cruz), and mouse anti-NDUFA9 (1:1,000, 459100, Invitrogen) antibodies. Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from GE healthcare. Signals on polyvinylidene difluoride (PVDF) membranes (Biorad) were detected using ECL prime (GE healthcare). Anti-9030617O03Rik rabbit polyclonal antibodies were generated against the full length mouse 9030617O03Rik peptide (1:1,000).