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. 2016 Apr 25;7(23):34759–34772. doi: 10.18632/oncotarget.8976

Figure 4. Deubiquitinating activity of USP19 on CORO2A.

Figure 4

A. and B. Cell lysates from HEK 293T cells which transfected with His-tagged ubiquitin, Flag-tagged CORO2A and/or Myc-tagged USP19 and the catalytic mutant USP19 (C506S) were subjected to in vitro ubiquitination and deubiquitination assay with Ni-NTA beads. MG132 (2.5μM) as a proteasome inhibitor was treated for 6 h before cell harvest. Western blotting was performed with indicated antibodies. A, The ubiquitination level of CORO2A was increased when the cells were treated with MG132 (lane 4), a proteasome inhibitor. B, The overexpression of USP19, but not the catalytic mutant USP19 (C506S), dramatically reduced the ubiquitination level of CORO2A (lanes 4 and 5). C. In vivo ubiquitination and deubiquitination assays were performed to identify the specific deubiquitinating activity of USP19 toward CORO2A. Myc-tagged USP19 and the catalytic mutant USP19 (C506S) were overexpressed in the 293T cells, and the cell lysates were used for immunoprecipitation with an anti-ubiquitin antibody. Lane 1 shows the ubiquitination of CORO2A. D. and E. USP19 and the catalytic mutant USP19 (C506S) were transfected into 293T cells by dose dependent manner (0, 0.3, 0.6, 0.8, and 1.0 μg) and cell lysates were analyzed with indicated antibodies. F. USP19 siRNA was transfected into 293T cells by dose dependent manner (0, 25, 50, 100, and 150 nM), and the expression level of USP19 and CORO2A was detected by anti-USP19 and anti-CORO2A antibodies. D and F, Statistical data are presented as a means (n=3, *p<0.05). G. Lysates from cells which respectively transfected with USP19 siRNA, HA-tagged Ubiquitin, and Flag-tagged CORO2A were subjected to the ubiquitination assay.