ω agatoxin iva

The chemical reagents used in this study are as follows: 100 μM nifedipine (Sigma-Aldrich, St. Louis, MO; N7634); 500 μM gadolinium chloride (Sigma-Aldrich, G7532); 10 μM SKF96365 (Sigma-Aldrich, S7809); 1 M EGTA (Sigma-Aldrich, E4378); 500 μM lanthanum chloride (Sigma-Aldrich, L4131); 2 μM ω-Agatoxin IVA (Tocris, Bristol, UK; 2799); 2 μM SNX 482 (Tocris, 2799); 10 μM ω-Conotoxin GVIA (Alomone Labs, C-300); 10 μM U0126 (Sigma-Aldrich, U120); 50 mM potassium chloride (Sigma-Aldrich, P3911); 100 nM AP24534 (Tocris, 4274); 25 μM trifluoperazine dihydrochloride (Sigma Aldrich, T8516).
The following plasmids were purchased from Addgene: pGP-CMV-GCaMP6s was a gift from Douglas Kim (Addgene plasmid # 40753)^{33 (link)}; pGP-CMV-GCaMP6s-CAAX was a gift from Tobias Meyer (Addgene plasmid # 52228)^{34 (link)}; AAV-CAG-GFP was a gift from Karel Svoboda (Addgene plasmid # 28014)^{35 (link)}. The generation procedures for ERK1-dTomato construct were described previously^{36 (link)}. AAV-CAG-GCaMP6s-CAAX was cloned by replacing the GFP sequence in the AAV-CAG-GFP vector with GCaMP6s-CAAX PCR amplicon flanked BamHI/HindIII restriction sites. pHelper, and pAAV-RC1 plasmids were purchased from Cell Biolabs. RaichuEV-HRas FRET biosensor was kindly gifted from Dr. M. Matsuda (Kyoto University).

PD was purchased from ChemFaces (Wuhan, Hubei, China). Dantrolene, bafilomycin A1, dl-TBOA, CGP37157, PD98059, GF109203X, H89, ω-conotoxin GVIA and ω-agatoxin IVA were obtained from Tocris Cookson (Bristol, UK). 3′,3′,3′-Dipropylthiadicarbocyanine iodide (DiSC3(5)) and Fura-2-AM were purchased from Invitrogen (Carlsbad, CA, USA). EGTA). Nicotinamide adenine dinucleotide phosphate (NADP⁺), glutamate dehydrogenase (GDH), sodium dodecyl sulfate (SDS) and all other chemical reagents were supplied by Sigma-Aldrich Co. (St. Louis, MO, USA).
Sucrose buffer: 320 mM sucrose, 10 mM HEPES, pH 7.4; HEPES buffer medium (HBM): 10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 5 mM NaHCO₃, 1.2 mM NaH₂PO₄ and 10 mM glucose and, pH 7.4. Radioimmunoprecipitation assay buffer (RIPA): 50 mM NaCl, 1.0% IGEPAL^® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0 (Sigma-Aldrich Co., St. Louis, MO, USA). Tris-buffered saline (TBS-T): 20 mM Tris, 150 mM NaCl, 0.1% (w/v) Tween^® 20 detergent.