HEK-293 cells were stably transfected with human influenza hemagglutinin (HA)-PTHR (HEK-293R) [14 (link)] and cultured in Dulbecco’s modified Eagle’s medium/F-12 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.75 mg/ml geneticin. HEK-293R cells express 6.0 × 105 PTHR/cell, with an average Kd of 14.2 nM. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2, 95% air. Cells were pre-incubated for 15 min with different concentrations of H2O2 (1–500 μM) followed by stimulation with 10 nM–1 μM [Nle8,18,Tyr34]-PTH(1-34) (hereafter NNT-PTH) (Bachem, Torrance, CA), an oxidant-resistant PTH peptide.
For transient transfections, HEK-293R cells were grown on poly-D-lysine-coated glass 25-mm coverslips for 12 h prior to transfection with FuGENE 6 (Roche Applied Science) in complete medium. After 24 h, coverslips in HEPES/ bovine serum albumin buffer (pH=7.4) (HEPES buffer containing 0.1% (w/v) BSA) were transferred to an Attofluor chamber (Invitrogen, Carlsbad, CA) for live-cell imaging.
For transient transfections, HEK-293R cells were grown on poly-D-lysine-coated glass 25-mm coverslips for 12 h prior to transfection with FuGENE 6 (Roche Applied Science) in complete medium. After 24 h, coverslips in HEPES/ bovine serum albumin buffer (pH=7.4) (HEPES buffer containing 0.1% (w/v) BSA) were transferred to an Attofluor chamber (Invitrogen, Carlsbad, CA) for live-cell imaging.