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Fig. 2.
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Next, we tested the ability of PRAJA1 to ubiquitinate the three core PRC2 members, EZH2, SUZ12, and EED in vitro. Recombinant PRAJA1 readily generated high molecular weight adducts of all three PRC2 proteins in an E2- and RING finger-dependent manner [ Fig. 2C(i)–(iii)] suggesting that it can target the three PRC2 members for degradation. Indeed, as can be seen in the experiment depicted in Fig. 2D(i)–(iii), PRAJA1 dramatically enhances the degradation of PRC2 subunits in a cell free assay in a RING finger- dependent manner.
3.3. PRAJA1 ubiquitinates the PRC2 proteins in DZNep and targets them for degradation
Fig. 3.
PRAJA1 affects the steady-state of EZH2 in cells. (A) MCF-7 cells were transiently transfected with cDNAs coding for HA-tagged EZH2 in the presence of Flag-tagged empty vector (EV), Flag-tagged TRIM47, PRAJA1, or ΔPRAJA1 in duplicates. (B) MCF-7 cells were transiently transfected with cDNAs coding for HA-tagged EZH2 in the presence of Flag-tagged EV or Flag-tagged TRIM47, PRAJA1, or ΔPRAJA1. 24h after transfection, cells were treated with DMSO or with 20 μM MG132 for 6 h as indicated. (C) MCF-7 cells were transiently transfected with cDNAs coding for HA-tagged EZH2 in the presence of Flag-tagged EV or Flag-tagged TRIM47, PRAJA1, or ΔPRAJA1. 48h after transfection, cells were treated with CHX for the indicated times. Proteins were resolved by SDS–PAGE and visualized after Western blot by the appropriate antibodies.