TMEM16 Protein Expression in HEK293 Cells

HEK293T cells (ATCC CRL-1573) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10 U ml⁻¹ penicillin, 0.1 mg ml⁻¹ streptomycin (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), and 10% FBS (Sigma-Aldrich) in a humidified atmosphere containing 5% CO₂ at 37 °C. HEK293S GnTI⁻ cells (ATCC CRL-3022) were maintained in HyClone HyCell TransFx-H medium (GE Healthcare) supplemented with 10 U ml⁻¹ penicillin, 0.1 mg ml⁻¹ streptomycin, 4 mM L-glutamine, 0.15% poloxamer 188 (Sigma-Aldrich), and 1% FBS in an atmosphere containing 5% CO₂ at 185 rpm at 37 °C. The ac splice variant of mouse TMEM16A (UniProt ID: Q8BHY3), mouse TMEM16B (UniProt ID: Q8CFW1), or mouse TMEM16F (UniProt ID: Q6P9J9) bearing a 3C cleavage site, a Venus YFP, a Myc tag, and a Streptavidin-binding peptide (SBP) downstream of the open reading frame in a modified pcDNA3.1 vector (Invitrogen) were used as described previously^{21 (link),31} . Mutations were introduced using a modified QuikChange method^{58 (link)} and were verified by sequencing.