Pcdna3 nedd4 2 flag

HEK cells and HEK cells stably co-expressing Nav1.5 and GFP were cultured in DMEM (Thermo Fisher) supplemented with 10% FBS. Transient transfections of HEK cells were performed for 48 h using Lipofectamine 2000 (Thermo Fisher) and following the manufacturer's instructions. Typically, we transfected 200 ng of pENTR-CMV-LITAF-HA-hrGFP (or pENTR-CMV-hrGFP), 200 ng of pcDNA3-NEDD4-2–FLAG, 200 ng of pcDNA3, and 1.8 µl of Lipofectamine 2000 per 12-well (Figs. 5, C and D, and 6B) or 500 ng of pRK5-HA-Ubiquitin-WT, 500 ng of pENTR-CMV-LITAF-FLAG-hrGFP (or pENTR-CMV-hrGFP), 500 ng of pcDNA3-Nedd4-2-FLAG, 1.5 µg of pcDNA3 (Invitrogen), and 7.2 µl of Lipofectamine 2000 per 6-cm dish (Fig. 6A). For transient transfections of HEK cells stably expressing Nav1.5, we generally used 1600 ng of pENTR-CMV-LITAF-HA-hrGFP (or pENTR-CMV-hrGFP), 1600 ng of pcDNA3-NEDD4-2–FLAG (or pcDNA3), 800 ng of pENTR-CMV-hrGFP, 270 ng of pDsRed-C1, and 10 µl of Lipofectamine 2000 (Figs. 2, B–D; 3A; and 4, A and B).