Glow max instrument

We analyzed a 766-bp fragment of the KCNJ18 promoter upstream of exon 1 for promoter activity. Wild-type or mutant KCNJ18 promoter constructs were generated by cloning a 766-bp polymerase chain reaction (PCR) fragment from the patient's genomic DNA containing KpnI/NcoI linkers into the corresponding sites of pGL3-Basic (Promega). Transient transfection assays in HEK293 cells were performed in six-fold reactions using HeLafect (OZ Biosciences) with 0.5 µg KCNJ18 pGL3 construct and 0.1 µg internal control plasmid pcDNA3.1 CMV-LacZ (Invitrogen). Cells were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 µg/mL). Forty-eight hours after transfection, the cells were harvested and cellular luciferase expression was measured using the luciferase assay system (Promega) on a Glow-Max—instrument (Promega). The assay was replicated 8 times in independent transfection experiments. Site-directed mutagenesis using the Gene Tailor Site-Directed Mutagenesis System (Invitrogen) was used to validate functional mutations from luciferase assays. For this purpose, the deleterious effect of the mutation was restored by introduction of the corresponding normal base into the mutant KCNJ18 promoter construct.

HS5 cells were transiently transfected with a Notch reporter plasmid pNL2.1 carrying a 6xCSL Notch responsive element^{26 (link)} and with the vector constitutively expressing the firefly luciferase upon the thymidine kinase promoter (pGL4.54[luc2/TK]). After 24 hours (h), HS5 cells were cultured alone or placed in co-culture with scrambled (Scr) or Jagged1 and Jagged2 knockdown (J1/2KD) HMCL and incubated for 24 h. Luciferase activity was measured using Nano-Glo^® Dual-Luciferase^® Reporter assay kit (Promega) on the Glowmax instrument (Promega).