Anti ip3r1

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×).

Twenty to thirty micrograms of total or mitochondrial protein was separated on SDS-polyacrylamide gel and electro-blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were saturated with blocking buffer for 1 h at room temperature and incubated overnight at 4°C with monoclonal mouse anti-VDAC (1:1000, Abcam), anti-OXPHOS (1:5000, Abcam), anti-IP3R1 (1:1000, Santa Cruz), anti-NDUFA13 (1:1000, Abcam), anti-GAPDH (1:10 000, Abcam) or with polyclonal rabbit anti-MCU (1:500, Abcam), anti-MICU1 (1:500, Thermo Fisher), anti-MICU2 (1:500, Sigma Aldrich), anti-PDH-E1α (1:1000, Abcam), anti-PDHE1α phosphor Ser 293 (1:1000, Abcam), anti-PDHE1α phosphor Ser 300 (1:1000, Millipore), anti-PDHE1α phosphor Ser 232 (1:1000, Calbiochem), anti-PDK4 (1:1000 Novus Biotech), anti-GRP75 (1:1000, Santa Cruz), anti-SIGMA1R (1:1000, Cell Signaling), anti-MFN2 (1:1000, Abcam), and anti-Hsp60 (1:1000, Abcam). Hsp60 and GAPDH were used as loading controls. All immunoblots were developed and quantified using the Odyssey infrared imaging system (LICOR Biosystems) and infrared-labeled secondary antibodies. Band intensities were quantified with ImageJ.

Lysates from primary melanocytes were purchased from Sciencell Research Laboratories (Carlsbad, CA) and NCI-60 validated human melanoma cell lines obtained from the University of Hawaii Cancer Center included SK-Mel2, SK-Mel28, and MALME-3M. These cell lines were cultured in RPMI media with 10% fetal bovine serum and 1% Antibiotic-Antimycotic (all from GIBCO/Thermo Fisher). Primary antibodies for western blots included rabbit monoclonal anti-SELENOK (Epigemonics, Inc., custom antibody), anti-IP3R1 (Santa Cruz Biotechnology, sc-271197), anti-GAPDH (Santa Cruz Biotechnology, sc-47724), anti-Prom1 (Cell Signaling, 58605). Antibodies from Abcam included anti-TRP2 (ab74073), anti-calcineurin A and B (ab137335, ab154650), and anti-calmodulin (ab 105498, respectively). Western blot secondary antibodies were purchased from Li-Cor Technologies and immunofluorescence secondary Alexafluor595 antibody from Thermo. The transgene encoding full-length SELENOK in pcDNA3.1+ (Invitrogen) has been previously described [11 (link)].