Ip3r3

For immunoblotting and co-immunoprecipitation: Actin (Santa Cruz, ♯sc-47778; 1:5000), ATF6 (Cell Signaling Technology, ♯65880; 1:1000), BCL-2 (AbCam, ♯ab182858; 1:500), BCL-XL (Cell Signaling Technology, ♯2764; 1:1000), Cleaved-Caspase-8 (Cell Signaling Technology, ♯8592; 1:1000), Caspase-3 (Cell Signaling Technology, ♯9665; 1:1000), Caspase-9 (Cell Signaling Technology, ♯9508; 1:1000), CHOP (Cell Signaling Technology, ♯2895; 1:250), GFP (AbCam, ♯ab13970; 1:2000), GRP78 (Cell Signaling Technology, ♯3177; 1:1000), FLAG-HRP (Sigma Aldrich, ♯A8592; 1:4000), HA-HRP (Roche, ♯11867423001; 1:2000), IP₃R1 (Thermo Fischer, ♯PA1-901; 1:1000), IP₃R1 & IP₃R2 [previously described⁷⁷ ; 1:1000], IP₃R3 (BD Biosciences, ♯610312; 1:1000), KDEL (AbCam, ♯ab12223; 1:2000), MCL-1 (Cell Signaling Technology, ♯5453; 1:1000), Nicastrin (BD Biosciences, ♯612290; 1:1000), PARP (Cell Signaling Technology, ♯9542; 1:1000), SERCA2 (Cell Signaling Technology, ♯4388; 1:1000), STIM1 (Cell Signaling Technology, ♯5668; 1:1000).
For immunofluorescence: DAPI (Thermo Fischer, ♯D1306; 1 µg/ml), HA (Cell Signaling Technology, ♯3724; 1:500), BAP31 (Enzo Life Sciences, ♯ALX-804-601-C100; 1:250).
For proximity ligation assay: HA (Cell Signaling Technology, ♯3724; 1:500), HA (Enzo Life Sciences, ♯ENZ-ABS118-0200; 1:200), IP₃R1 (Thermo Fischer, ♯PA1-901; 1:200), IP₃R3 (BD Biosciences, ♯610312; 1:200).

From Cell Signaling Technology: Phospho-α1-AMPK (Thr172), α1-AMPK, IP3R-1, p53, acetyl-p53, cortactin, Drp1. From BD Laboratories: β-tubulin, IP3R-3. From Sigma: MCU, acetyl-cortactin. From Abcam: Tomm20. Secondary antibodies conjugated with peroxidase were purchased from GE Healthcare Life Science. TMRE, Fluo-4, mitotracker, BAPTA-AM, BCECF-AM, DAPI, and secondary antibodies conjugated with Alexa 488, 546, 633 were from Molecular Probes. Chemicals: methyl-pyruvate, FCCP, oligomycin, rotenone, nicotinamide (NMN), latrunculin B (LatB), cytochalasin D (CytD), and Hanks’ balanced salt solution were purchased from Sigma. EX527 and compound C were purchased from Tocris Bioscience. Xestospongin B was extracted and purified from the marine sponge Xestospongia exigua as described before (Jaimovich et al., 2005 (link)).

Cells lysates were obtained by incubating cells directly with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. After ultrasonicating 5 times (5 s each), lysates were heated at 100 °C for 10 min. Proteins were separated on 6% SDS-PAGE gel (for IP₃R expression) or 8% SDS-PAGE gel (for α2δ1, α2δ2, and SERCA3 expression) and transferred to a 0.45-μm polyvinylidene difluoride membrane (Millipore). Membranes were blocked with 5% bovine serum albumin (for IP₃R expression) or 5% nonfat dry milk (for α2δ1, α2δ2, and SERCA3 expression) and incubated with primary antibody overnight at 4 °C. Primary antibodies against IP₃R1 (Abcam, 1:500), IP₃R2 (Millipore, 1:50), IP₃R3 (BD Biosciences, 1:1000), α2δ1 (Abcam, 1:1000), α2δ2 (Sigma, 1:2000), SERCA3 (Abcam, 1:500), and tubulin (Sigma-Aldrich, 1:2000) were used.

Finely minced salivary glands were homogenized in a lysis buffer supplemented with protease inhibitor cocktail (Complete mini; Roche Diagnostics) for 16–20 strokes. After incubating on ice for 30 minutes, solubilized proteins were separated by centrifugation at 13000 rpm at 4℃ for 30 minutes. 10μg of protein lysate was loaded on 7.5%- 12% SDS- polyacrylamide gels. Subsequently, the proteins were transferred to PVDF membranes at a voltage of 35V at 4°C overnight. The membrane was blocked with 5% non-fat skimmed milk in TBST (50 mM Tris-HCl, pH 7.5 with 0.1% Tween20) at RT for 1 hour and subsequently incubated with primary antibodies overnight at 4°C (Actin (Millipore Sigma; A2228; 1:10000), IP₃R2 (Antibody Research Corporation; 1:1000), IP₃R3 (BD Transduction Laboratory; Cat. 610313; 1:1000), TMEM16a (Abcam; ab84115; 1:1000)). After being washed with 0.1% TBST, the membranes were incubated with secondary antibodies at RT for 1 hour (Goat anti-rabbit IgG (H&L) (Invitrogen; SA535571; 1:10000), Goat anti-mouse IgG (H&L) (Invitrogen; SA535521; 1:10000)). Protein band intensity from western blotting was quantified by FIJI. The relative ratio of DMXAA-treated/ vehicle control was calculated in Excel. Lastly, graphical generation and statistics were performed with a t-test using Prism (GraphPad) as indicated in the figure legends.

The following antibodies were used: β3 tubulin (Santa Cruz Biotechnology, Dallas, TX, USA, #80016), Actin (Sigma-Aldrich, St. Louis, Missouri, USA, #A4700), APP 6E10 (BioLegend, San Diego, CA, USA, #803001), APP Y188 (Abcam, Cambridge, UK, #ab32136), Drp1 (BD Bioscience, San Jose, CA, USA, #611112), GAPDH (Enzo LifeScience, Exeter, UK, #ADI-CSA-335-E), IP3R3 (BD Biosciences, San Jose, CA, USA, #610312), LC3B (Cell Signalling, Danvers, MA, USA, #3868; Novus Biologicals, Centennial, CO, EUA, #NB100-2220), Mfn1 (Santa Cruz Biotechnology, #SC50300), Mfn2 (Abcam, #Ab56889), Opa1 (BD Bioscience, San Diego, CA, USA, #612606), SQSTM1/p62 (Cell Signalling,#5114), TIM23 (BD Biosciences, #611223), TOM20 (Santa Cruz Biotechnology, #sc-11415), TOM70 (Santa Cruz Biotechnology, #sc-366282) and VDAC1 (Abcam, #Ab14734).