Ryanodine

Tetrodotoxin, phentolamine, iberiotoxin, NS-1619, Y-27632, apamin, NS-309, and ryanodine were purchased from Abcam (Cambridge, MA, USA). Atropine, propranolol, suramin, nisoldipine, and SKF-96365 were purchased from Sigma-Aldrich (St. Louis, MO USA). Bisindolylmaleimide 1 and H₂O₂ were obtained from Cayman Chemical (Ann Arbor, MI, USA) and Fisher Scientific (Pittsburg, PA, USA), respectively.
NS-1619, nisoldipine, NS 309, and SKF-96365 were dissolved in dimethylsulphoxide (DMSO). ryanodine was dissolved in 100% ethanol. All other drugs were prepared with water. Final ethanol and DMSO concentrations in the bath solution did not exceed 0.1% and were shown not to affect spontaneous contractions or bladder function in previous reports [35 (link)–37 (link)].

TM, CPA, TG, and XeC were obtained from Cayman Chemical, Ann Arbor, MI. Dan was from Sigma-Aldrich, St Louis, MO. Ryanodine (Ry) was from Abcam, Waltham, MA. RNeasy mini kit for RNA extraction was from Qiagen, Germantown, MD. Superscript RT II was from Invitrogen, Carlsbad, CA. SYBR Green PCR master mix was from Applied Biosystems, Bedford, MA. Primers for qRT-PCR were from Integrated DNA Technologies, Coralville, Iowa. Small interfering RNAs (siRNAs) were from ThermoFisher scientific, Waltham, MA. The catalog and lot numbers of these materials can be found in Table S1.

H9c2 (2–1) rat cardiomyoblast cell lines were obtained from American Type Culture Collection (Manassas, VA, USA). [³H]-Ryanodine was obtained from New England Nuclear (Milan, Italy). Ryanodine was purchased from Abcam, UK. Unless otherwise specified, all reagents were obtained from Sigma-Aldrich (St. Louis, MO, USA). Solvents for HPLC–MS/MS measurements were HPLC-grade, and the other chemicals were reagent-grade.

Hanks’ Balanced Salt Solution (HBSS) was prepared by supplementing 20 mM HEPES buffer and adjusted to pH 7.4 (sHBSS). Hanks’ 0-Ca²⁺ solution (0-Ca²⁺ sHBSS) was prepared with no-Ca²⁺ HBSS supplemented with 1 mM Na₂H₂-EGTA, 20 mM HEPES and 0.9 mM MgSO₄. Oregon Green 488 BAPTA-1 a.m. was purchased from Life Technologies (Grand Island, NY, United States). Ryanodine was purchased from Abcam Inc (Cambridge, MA, United States), other reagents were purchased from either Sigma-Aldrich (St. Louis, MO, United States) or Thermo Fisher Scientific (Pittsburgh, PA, United States).

Murine melanoma cell line B16 and OVA-B16, murine hepatocarcinoma cell line H22, and murine macrophage cell line Raw264.7 were purchased from China Infrastructure of Cell Line Resources (Beijing, China) and cultured with RMPI-1640 (Life Technologies) or high glucose-Dulbecco’s modified Eagle’s medium (Corning) medium supplemented with 10% feral bovine serum (FBS) (Gibco, Australia) at 37 °C with 5% CO₂. Cells were tested for mycoplasma detection, interspecies cross-contamination, and authenticated by isoenzyme and short tandem repeat analyses in Cell Resource Centre of Peking Union Medical College before the beginning of the study and spontaneously during the research. Chloroquine, OVA257-264, OVA323^-339, and CY were purchased from Sigma-Aldrich. SB203580, JSH-23, Baf, IPI-549, 1400W 2HCl, and CsA were purchased from Selleck. Anti-CD3 (145^-2C11), anti-CD8 (53-6.7), anti-CD25 (PC-61.5.3) mouse neutralizing antibody, and isotype control (N/A) were purchased form Bio X Cell. Purified anti-mouse Ly-6G/Ly-6C (Gr-1) antibody (RB6-8C5) and rat IgG2bκ isotype control were purchased from BioLegend. The macrophage depletion reagent, clodronate liposomes, was purchased from FormuMax Scientific USA. Ryanodine and CGP37157 were purchased from Abcam.

Human embryonic kidney 293 (HEK293) cell line, plasmid pcDNA3, plasmid pcDNA5, Tris-HCl (pH 8.8), MgSO₄, Triton X-100, bovine serum albumin (BSA), dATP, dCTP, dGTP and dTTP (Amersham), DNA polymerase (Stratagene), QIA quick PCR Purification Kit, Flp-In T-Rex Core Kit (Invitrogen), phosphate-buffered saline (PBS) (137 mM NaCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄ and 2.7 mM KCl), hygromycin (Invitrogen), Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum, HEPES buffer (274 mM NaCl, 1.8 mM Na₂HPO₄ and 50 mM HEPES, pH 7.04), KRH (Krebs–Ringer–HEPES) buffer (125 mM NaCl, 5 mM KCl, 1.2 mM KH₂PO₄, 6 mM glucose, 1.2 mM MgCl₂ and 25 mM HEPES, pH 7.4), CaCl₂, fluo 3, sulfinpyrazone, Fura-2 acetoxymethyl ester (Fura-2 AM), tetracycline, pluronic F-127, caffeine, EGTA, EDTA, Tris, CHAPS, soybean phosphatidylcholine, DTT, benzamidine, leupeptin, pepstatin A, aprotinin, PMSF, L-glutamine, penicillin, nonessential amino acids, Tween-20, skimmed-milk powder, anti-RyR antibody (34c), anti-mouse IgG (H&L) antibodies, enhanced chemiluminescence kit (Pierce), [³H]ryanodine (PerkinElmer), ryanodine (Abcam).