Cloning and Mutagenesis of Human USP19 and Coro2A

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).

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Lim K.H., Choi J.H., Park J.H., Cho H.J., Park J.J., Lee E.J., Li L., Choi Y.K, & Baek K.H. (2016). Ubiquitin specific protease 19 involved in transcriptional repression of retinoic acid receptor by stabilizing CORO2A. Oncotarget, 7(23), 34759-34772.

Publication 2016

pcDNA3-Myc Opti-MEM RNAimax

Cells Human Nagase Sirna Site directed mutagenesis Ubiquitin Vector

Corresponding Organization : CHA Bundang Medical Center

Other organizations : CHA University

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Variable analysis

independent variables

Different concentrations of USP19 siRNA (0.5, 1 and 1.5 nmol)

dependent variables

Not explicitly mentioned

control variables

Myc-tagged USP19 (C506S) mutant generated by site-directed mutagenesis
His-tagged ubiquitin cloned as previously described
Coro2A subcloned into a pCS4-Flag vector

controls

Positive control: Not specified
Negative control: Not specified

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