Transient Transfection of Mutant AP2S1 in HEK Cells

Wild-type and mutant pBI-CMV4-AP2S1 expression constructs were generated, as described(12 (link)) and transiently transfected into human embryonic kidney (HEK)-293 cells stably expressing CaSR (HEK-CaSR)(7 (link),8 (link)) using Lipofectamine 2000 (Life Technologies). The bidirectional pBI-CMV4 cloning vector was used because it facilitated the co-expression of AP2σ and red fluorescent protein (RFP).(12 (link)) Site-directed mutagenesis was used to generate the mutant AP2S1 constructs using the Quikchange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and gene-specific primers (Sigma-Aldrich), as described.(25 (link)) Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM)-Glutamax media (ThermoFisher) with 10% fetal bovine serum (Gibco) and 400 μg/mL geneticin (ThermoFisher) at 37 °C, 5% CO₂. Transfection was confirmed by Western blot analyses and by visualizing RFP fluorescence using an Eclipse E400 fluorescence microscope with a Y-FL Epifluorescence attachment and a triband 4,6-diamidino-2-phenylindole-FITC-Rhodamine filter, and images taken using a DXM1200C digital camera and NIS Elements software (Nikon), as described.(12 (link))