Fluo 4 am

Dulbecco's modified Eagle's medium (DMEM), Dilauroyl‐ phosphatidylcholine, and RN‐1747 were purchased from Wako Pure Chemicals (Osaka, Japan). Fetal calf serum (FCS), penicillin‐ streptomycin solution, ketoconazole, and arachidonic acid sodium salt were purchased from Sigma Chemical (St. Louis, MO). 5,6‐, 8,9‐, 11,12‐, and 14,15‐EET, 5‐, 8‐, 9‐, 11‐, 12‐, 15‐, 16‐, 17‐, 18‐, 19‐, and 20‐HETE, and capsazepine were purchased from Cayman Chemical Co. (Ann Arbor, MI). Antibodies against CYP2C11, 2C23, 2E1, 4A2, sEH, NADPH‐cytochrome P450 reductase, and β‐actin were prepared as described previously.16, 17, 18 β‐NADPH was purchased from Oriental Yeast Co. LTD (Tokyo, Japan), horse serum was from Equitech‐Bio (Kerrville, TX), and nerve growth factor (NGF) was form Alomone Labs (Jerusalem, Israel). HC067047 and Fura‐2 AM were from Abcam plc (Cambridge, UK). Fluo‐4AM was from AAT Bioquest (Sunnyvale, CA).

Calcium imaging was carried out at room temperature within 6 h after isolation of cardiomyocytes from rats in the control group (Zhang et al., 2020 (link)). Ca²⁺ transients in the cardiomyocytes were recorded using an LSM-710 laser-scanning confocal microscope (Carl Zeiss, Inc, Germany) with a ×40 magnification, 1.3 numerical aperture oil immersion objective, and axial resolutions of 1.5 μm. Fluo-4 AM: Fluo-4AM+8μlDMSO+2μlF-127 was first prepared and then diluted 1,000 times using 1 ml DMem+1 μl Fluo-4. Cardiomyocytes were then incubated with 500–10,002 μl Fluo-4 AM (AAT Bioquest, Inc. Sunnyvale, CA, United States) for 20 min at 37°C and recorded in normal Tyrode’s solution. Fluo-4 was excited at 488nm, followed by measurement of the fluorescence emission at 505 nm. Images were acquired in the line-scan (X-T) mode with 512 pixels (pixel intervals 0.15 μm) per line at a rate of 3 ms per scan. The Ca²⁺ transients were then analyzed using a modified version of the MATLAB program, and their fluorescence emission intensity was expressed as F/F0, where F0 was the basal fluorescence intensity level. The recording was performed at 35°C.

BmK venom was purchased from a domesticated scorpion farm (Kaifeng, China), where it was collected by electrical stimulation. Sephadex G-50 and CM-Sephadex C-50 were purchased from Pharmacia Fine Chemicals (Uppsala, Sweden). Acetonitrile (HPLC grade) was from Tedia (Cincinnati, OH, USA). Dialysis membrane (500 Da cut off) was a product of Minnesota Mining and Manufacturing (St. Paul, MN, USA). Trypsin, L-glutamine, fetal bovine serum, Neurobasal medium, Hoechst 33,342, and anti-MAP2 primary antibody were obtained from Life Technology (Grand Island, NY, USA). Primary antibodies against Akt, phosphorylated (p)-Akt, were obtained from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies and NewBlot Nitro Stripping Buffer were from LI-COR Biotechnology (Lincoln, NE, USA). Trifluoroacetic acid, cytosine arabinoside, poly-L-lysine, N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES), GW 441756, and all inorganic salts were obtained from Sigma-Aldrich (St. Louis, MO, USA). Tetrodotoxin was purchased from Tocris Bioscience (Ellisville, MO, USA). The Ca²⁺-specific fluorescent dye Fluo-4/AM was obtained from AAT Bioquest (Sunnyvale, CA, USA). NGF ELISA kit was purchased from Kete Biological Technology Co., Ltd. (Nanjing, China).