The SIM of PML is not required for the interaction with RNF4 but is necessary for PML ubiquitination in response to As₂O₃.

<p>(A) Co-immunoprecipitation of RNF4 and PML isoforms and mutant. HEK293 cells were co-tranfected with a construct encoding Flag-RNF4 and PMLIII, PMLVI or PMLIII-SIM. As controls, cells were transfected with PMLIII or RNF4 alone. Two days later, the different cell extracts were immunoprecipitated with an anti-Flag antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044949#s4" target="_blank">Materials and Methods</a> and samples were analyzed by Western blot using anti-PML and anti-Flag antibodies. (B) Ubiquitination of PML in response to As₂O₃. PML^−/− MEFs were transiently transfected with the empty vector (EV) or PMLIII (left panel) or contructs encoding PMLIII, PMLIII-SIM or PMLVI (right panel). Two days later, they were treated with As₂O₃ for 1 h. The cell extracts were immunoprecipitated with rabbit anti-PML antibody and the immunoprecipitate was analyzed by Western blot using anti-ubiquitin and anti-PML antibodies.