Adenoviral Expression of KCNH6 Mutants

Mouse KCNH6 and Munc-18-1 cDNAs were derived from MIN6 cells. Point and deletion mutants were generated using a standard PCR-based mutagenesis strategy and were verified by DNA sequencing. The sequences of the primers used are listed in Supplementary Table 2. These cDNAs were subcloned into pcDNA3-HA, pcDNA3-FLAG vector (Invitrogen) or mCherry-C1 vector (Clontech) as described previously [24 (link)]. Insulin-EGFP was generated as described previously [25 (link)]. To generate recombinant adenoviruses, KCNH6 WT and KCNH6 R246A/T248A/L250A (3A) mutant were inserted into pENTR-3C (Invitrogen) and were transferred into pAd/CMV by LR Clonase recombination (Invitrogen), which co-produces red fluorescent protein (Cherry) to allow identification of transfected cells. To express an exogenous protein, HEK293A cells were transfected with the plasmids using Lipofectamine 3000 reagent (Invitrogen), whereas MIN6 cells were infected with adenoviruses.