Heat inactivated fetal bovine serum

Human fibrosarcoma HT1080 cells (KCLB no. 10121), human prostate adenocarcinoma PC-3 cells (KCLB no. 21435), and human gastric carcinoma AGS cells (KCLB no. 21739) were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI 1640 (Cellgro, Manassas, VA, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Cellgro) and penicillin (100 U/mL)/streptomycin (100 μg/mL) (Cellgro) at 37°C in a humidified 5% CO₂ incubator. Murine hepatocytes were isolated using a perfusion system with some modification and incubated as described previously [19 (link)].

C7/10 Aedes albopictus cells were grown at 28 °C under 5% ambient CO₂ in 1× Minimal Essential Media (Corning, Corning, NY, USA) supplemented with 10% heat-inactivated fetal bovine serum (Corning, Corning, NY, USA), 1% L-glutamine (Corning, Corning, NY, USA), 1% non-essential amino acids (Corning, Corning, NY, USA), and 1% antibiotic–antimycotic solution (Corning, Corning, NY, USA). Vertebrate baby hamster kidney fibroblast or BHK-21 cells as well as human embryonic kidney epithelial or HEK-293 cells were grown at 37 °C under 5% ambient CO₂ in the same 1× Minimal Essential Media (Corning, Corning, NY, USA) as the C7/10 cells.

Chinese hamster lung fibroblasts were obtained from the American Type Culture Collection. A stable HEK cell line expressing human (h) Na_v1.1 was a gift from GlaxoSmithKline. CHL and HEK‐hNa_v1.1 cells were maintained at 37°C and 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 5% heat‐inactivated fetal bovine serum (Corning) and 100 U/mL penicillin/streptomycin (Gibco). Stably transfected CHL cells and HEK‐hNa_v1.1 cells were supplemented with 600 µg/mL G418 (Gibco). Stable β1‐V5‐2AeGFP or β1‐p.Arg85Cys‐V5‐2AeGFP cell lines were generated by transfecting 1 µg of cDNA with 5 µL of Lipofectamine 2000. About 48 h following transfection, cells were passaged into media containing 600 µg/mL G418 and incubated until individual, eGFP‐positive colonies were visible (approximately 7–10 days). Clonal colonies were isolated, expanded, and verified for β1‐V5 or β1‐p.Arg85Cys‐V5 expression via western blot with anti‐V5 antibody. For electrophysiological analyses, HEK‐hNa_v1.1 cells were transiently transfected with β1‐V5‐2AeGFP, β1‐p.Arg85Cys‐V5‐2AeGFP, or eGFP only (1 µg of cDNA with 5 µL of Lipofectamine 2000). Approximately 12 h following transfection, the cells were passaged at low density onto 35‐mm dishes. Cells were identified by eGFP fluorescence by an investigator blind to genotype.

Human HEC-1-B endometrial cells (ATCC strain HTB-113) were grown at 37°C in 5% CO₂. Cells were maintained in Eagle’s minimal essential medium (MEM [Corning]), supplemented with 10% heat-inactivated fetal bovine serum (Corning) and 50 to 100 IU/ml penicillin and 50 to 100 µg/ml streptomycin (Corning). Cells were split every 3 to 4 days and passaged at a 1:4 dilution. Twenty-four hours prior to infection, antibiotic-containing medium was aspirated, cells were washed with sterile PBS, and medium was replaced with antibiotic-free, serum-free MEM. At the time of infection the medium was aspirated and replaced with fresh antibiotic-free, serum-free medium supplemented with or without 1.25 mg/ml hyaluronic acid. All GBS infections were performed at an approximate MOI of 0.01 for 48 h between passages 7 and 14.

HEK293T cells were cultured at 37 °C 5% CO₂ in 100-mm dishes containing minimum essential medium (Gibco) supplemented with 1× GlutaMAX (Gibco) and 10% heat-inactivated fetal bovine serum (Corning). At 85% confluency, cells were transfected with HA2-KEAP1 and/or Myc3-NRF2 plasmid constructs using the Lipofectamine 2000 Transfection Reagent (Thermo Fisher Scientific). Twenty-four hours post-transfection, cells were treated with proteasomal inhibitor MG-132 (Sigma Aldrich). Cells were then lysed 4 h later with a IGEPAL CA-630 lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 50 mM NaF, 1 mM Na₃VO₄, 0.1% IGEPAL CA-630, 1 mM dithiothreitol [DTT], 1× protease inhibitor cocktail). Cell lysates were incubated with monoclonal anti-HA conjugated magnetic bead slurry (MBL) overnight at 4 °C on an inverting rotator. Lysates were washed three times in wash buffer (TBS, 0.1% IGEPAL CA-630, 1 mM DTT), and immunoprecipitated HA2-tagged KEAP1 was eluted by adding 1× Laemmeli buffer, boiling for 5 min, followed by physical separation from magnetic bead slurry using a magnetic rack (MBL). HA2-KEAP1 and/or Myc3-NRF2 proteins were visualized and quantified through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.