Pcdna3 myc

HeLa, HEK293, and MCF7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, GE Healthcare Life Sciences, Marlborough, MA, USA), supplemented with 10% (v/v) fetal bovine serum (FBS; Hyclone, GE Healthcare Life Sciences, UT, USA), and 1% (v/v) penicillin/streptomycin solution (PS; Hyclone, GE Healthcare Life Sciences, UT, USA) in a humidified CO₂ incubator. Full-length MED28, CDKs, ETS-1, E2F-1, NRF-1, and C/EBPβ cDNAs were PCR-amplified and subcloned into vectors pcDNA3-Flag, pcDNA3-Myc (Invitrogen, Carlsbad, CA, USA), pEXPR-Iba-105 (Iba Lifescience, Goettingen, Germany), or pEGFP-C1 (Clontech, Mountain View, CA, USA) vectors.

The KIAA0891 clone containing the coding region of human USP19 was kindly donated by Dr. Nagase at the KAZUSA Institute in Japan [40 (link)]. The coding region of human USP19 was subcloned into pcDNA3-Myc (Invitrogen, Carlsbad, CA, USA). Myc-tagged USP19 (C506S) mutant was generated by site-directed mutagenesis. Using a commercial clone (IMAGE clone: 3350035) containing the coding region of Coro2A, we subcloned Coro2A into a pCS4-Flag vector (Invitrogen, Carlsbad, CA, USA). His-tagged ubiquitin was cloned as previously described [41 (link)]. Different concentrations of USP19 siRNA (0.5, 1 and 1.5 nmol) were transfected into cells by using an Opti-MEM and RNAimax (Invitrogen, Carlsbad, CA, USA) mixture according to the manufacturer's instructions. The USP19 sense siRNA sequences were; 5′-GGA GGA GAU GGC AGU GGC A-3′. The anti-sense siRNA sequences were; 5′-UGC CAC UGC CAU CUC CUC C-3′ (UbiProtein Corp, Seongnam, Korea).

rEag1 (kindly provided by Dr. Olaf Pongs, Institute fur Neurale, Signalverarbeitung, Zentrum fur Molekulare Neurobiologie, Germany) and hERG1 (hERG1a; kindly provided by Dr. Gail A. Robertson, Department of Neuroscience, University of Wisconsin, USA) cDNAs were subcloned into the pcDNA3-myc (Invitrogen) and the pSP64-polyA vectors, respectively. To generate N-terminal chimeras between rEag1 and hERG1, compatible restriction sites were introduced in both rEag1 and hERG1 through silent or missense mutations by using the QuickChange site-directed mutagenesis kit (Stratagene). Chimera P: HindIII (in vectors) and PmeI#1 (rEag1-A135A & K137K; hERG1-K135F, D136K, & M137L). Chimera N: HindIII and PmeI#2 (rEag1-V215V, F216F, K217K, & T218L; hERG1-A408L). Eag-domain deletion (Δeag): double-digestion with HindIII and PmeI#1, followed by insertion of the tri-amino-acid linker sequence LAG. To generate the rEag1 and the hERG1 eag-domain fragments, a stop codon was introduced at the end of the eag domain in rEag1-WT and rEag1-chimera N, respectively. The rEag1 point mutations Y344C, Y344A, and Y344F were also generated by using the QuickChange site-directed mutagenesis kit. All constructs were subject to DNA sequencing verification.