Ptdtomato n1 vector

Zebrafish Ikk1 (Chuk) cDNA was purchased from Open Biosystems (Clone ID: 5914247, GE Dharmacon). Full-length (FL)-Ikk1 was amplified with Advantage 2 Taq Polymerase (Clontech) using forward primer 5′-GCTAGCTGTCAATATGGAGAAACCCCCT-3′ and reverse primer 5′-TAATGGATTTGGTACCGACAAACGCGCTGATTTA-3′. The amplified PCR products were cloned into pCR Blunt II-TOPO using the Zero Blunt^® TOPO^® PCR Cloning Kit (Life Technologies). To generate tdTomato fusion constructs, pCRTOPO-FL-Ikk1 and ptdTomato-N1 vector (Clontech) were digested with KpnI and NheI, and ligated. The Ikk1(C179A)–tdTomato variant was generated from FL-Ikk1–tdTomato using site-directed mutagenesis (GeneArt^® Site-Directed Mutagenesis PLUS Kit, LifeTechnologies). The constructs were subsequently cloned into a plasmid containing the zebrafish tp63 promoter (pT2KXIG_tp63:AcGFP, courtesy of Gromoslav Smolen, Harvard University, Cambridge, MA). First, GFP was removed via digestion with BamHI and NotI, and both sites were Klenow blunt-ended. The Ikk1–tdTomato plasmids were digested with SnaBI and SfoI and ligated into pT2KXIG_tp63 to generate tp63:FL-ikk1–tdTomato and tp63:ikk1(C179A)–tdTomato. HEK001 were transfected using Nanofectin (PAA, Q050-005) when 60% confluent in 8-chamber vessels. It was determined that 0.5 µg of DNA per 0.6 µl of transfection reagent was optimal.