Fig. 5 | Nature Communications

Fig. 5

From: Heterozygous deletion of chromosome 17p renders prostate cancer vulnerable to inhibition of RNA polymerase II

Fig. 5

RBX1 modifies POLR2A by the K63-linked ubiquitination. a RBX1 physically interacts with POLR2A. Immunoprecipitation (IP) and western blot analyses were performed using indicated antibodies. Normal IgG was used as a negative control for IP. b, c RBX1 overexpression (b) or knockdown (c) increases or decreases the level of ubiquitinated POLR2A, respectively. DU145 cells transfected with indicated expression vectors were treated with MG132 and ubiquitinated POLR2A was pulled down (IP) and subject to immunoblotting (IB) analysis. d, e RBX1-mediated ubiquitination acts on the lysine-63 (K63) residue of POLR2A. DU145 cells were transfected with the indicated ubiquitin expression vectors and treated with MG132. Cells were also treated with Dox to induce RBX1 knockdown in d, or ectopic Flag-RBX1 was expressed in the cells in e. Ubiquitinated POLR2A was pulled down (PD) with Ni-NTA Agarose and subject to immunoblotting (IB) analysis. f, g RBX1 knockdown (f) or overexpression (g) decreases or increases the level of lysine-63 (K63) ubiquitination of POLR2A, respectively. Equal amounts of cell lysates were analyzed by immunoprecipitation and IB assays as described above. Data are representative of three independent experiments

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