Fig 8.

Arkadia and RNF4 have independent STUBL functions. (A) Arkadia and RNF4 homodimerize but do not form heterodimers. HEK293 cells were transfected with Flag-Ark-wt or Flag-RNF4-wt constructs, and equal amounts of whole-cell lysates were subjected to GST pulldown experiments with immobilized GST, GST-RNF4, or GST-Ark-665-Cter. Arkadia and RNF4 proteins were detected by Western blotting with anti-FLAG antibody before (5%) and after pulldown of the lysate. Blots were stained with Ponceau S prior to immunostaining in order to control the amount of GST proteins in the experiment. (B) Arkadia and RNF4 are independently involved in arsenic-induced degradation of PML. HT1080-GFP-PML cells were transfected with a limiting amount of siRNA, siNT (60 nM), siArk (50 nM), or siRNF4 (10 nM) or with a combination of siArk (50 nM) and siRNF4 (10 nM). At 48 h after transfection, cells were exposed or not to arsenic for 1 h or 24 h. Whole-cell lysates were analyzed by Western blotting with anti-GFP, anti-Ark, anti-RNF4, and antiactin antibodies. Numbers to the left of the gels are molecular masses (in kDa).