Dmem and f 12

DEX was purchased from Sigma (St. Louis, MO). Recombinant human TNF, PTX3, mouse anti-human ERK1/2 mAb, affinity purified rabbit anti-phosphoERK1/2 (T202/Y 204) and ELISA kits for human PTX3 were purchased from R&D Systems (Minneapolis, MN). The p38 MAPK inhibitor, SB-203580[4-4-fluorophenyl)-2-(4-methyl-sulfinylphenyl)-5- (4’-pyridyl)-1H-imidazole], the p42/ p44 ERK inhibitor, U-0126 [1, 4-diamino-2, 3-dicyano-1, 4-bis (2-aminophenyl-thio) butadiene], and the PI3K inhibitor, wortmannin, were purchased from Calbiochem (Mississauga, Ontario, Canada). All cell culture media (DMEM and F-12), antibiotics (penicillin, streptomycin), trypsin-EDTA, and cell culture reagents were obtained from Invitrogen Life Technologies, and FBS was from HyClone Laboratories (Logan, UT). The anti-human smooth muscle actin antibody (Ab) was obtained from DakoCytomation. Alkaline phosphatase-conjugated streptavidin was purchased from Jackson ImmunoResearch Laboratories. Unless stated otherwise, all other reagents were obtained from Sigma-Aldrich.

HEK293 cells were maintained in DMEM and F‐12 (Invitrogen, Carlsbad, CA), supplemented with 0.05% glucose, 0.5 mM pyruvate, 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin (Invitrogen), and incubated at 37ºC with 5% CO₂. HEK293 cells stably expressing the human Nav1.6 channel (hereafter referred to as HEK‐Nav1.6 cells) were maintained similarly except for the addition of 500 μg/ml G418 (Invitrogen) to maintain stable Nav1.6 expression. Cells were transfected at 80%–90% confluence with equal amount (1 μg each) of plasmid pairs using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions. HEK‐Nav1.6 cells were washed and replated at very low density prior to electrophysiological recordings (Ali et al., 2016, 2018; Scala et al., 2018; Wadsworth et al., 2019).