Journal: Molecular and Cellular Biology

Article Title: Arkadia, a Novel SUMO-Targeted Ubiquitin Ligase Involved in PML Degradation

doi: 10.1128/MCB.01019-12

Figure Lengend Snippet: Arkadia is involved in arsenic-induced degradation of sumoylated PML. (A) Depletion of Arkadia inhibits arsenic-induced degradation of sumoylated PML. HT1080-GFP-PML cells were transfected with a nontargeting siRNA (siNT) and siRNA SMARTpool targeting Arkadia (siArk) or RNF4 (siRNF4). Cells were exposed to arsenic at the indicated times. Whole-cell lysates were analyzed by Western blotting with anti-Ark, anti-RNF4, anti-GFP, and antiactin antibodies. A high exposure (exp.) and a low exposure of the same anti-GFP Western blot are shown. (B) Depletion of Arkadia inhibits arsenic-induced degradation of PML nuclear bodies. HT1080-GFP-PML cells were transfected with siNT, siArk, or siRNF4. Cells were exposed to arsenic at the indicated times before fixation, permeabilization, and DAPI staining. GFP-PML (green) expression and cellular localization were then analyzed by GFP-based fluorescence with the same laser intensity for each condition and are represented alone or merged with DAPI (blue). Bars, 20 μm. (C) Quantification of the number of PML nuclear bodies visualized with anti-PML staining in 30 cells under the indicated conditions, as described for panel B. (D and E) The same experiments described for panels A and B, respectively, performed with two individual siRNAs targeting Arkadia, siArk(1), and siArk(2). Numbers to the left of the gels are molecular masses (in kDa).

Article Snippet: GFP-PML-IV-wt cloned into the pBabe-puro lentiviral vector (Cell Biolabs) was used for infection.

Techniques: Transfection, Western Blot, Staining, Expressing, Fluorescence

Journal: Molecular and Cellular Biology

Article Title: Arkadia, a Novel SUMO-Targeted Ubiquitin Ligase Involved in PML Degradation

doi: 10.1128/MCB.01019-12

Figure Lengend Snippet: Arkadia interacts with sumoylated PML through its SIMs. (A and B) Arkadia specifically interacts with SUMO-modified PML through its SIMs. HEK293 cells were transfected with the indicated Flag-Ark constructs together with HA-PML (A) or with GFP-PML-wt or GFP-PML-3KR mutated in the 3 SUMO consensus sites (B). Cells were treated or not with arsenic for 1 h. Flag immunoprecipates were analyzed by Western blotting using anti-Flag and anti-HA (A) or anti-GFP (B) antibody. The corresponding whole-cell lysates (input) were analyzed using anti-HA (A) or anti-GFP (B) antibody. #, nonspecific binding of unmodified GFP-PML under all conditions. Numbers to the left of the gels are molecular masses (in kDa). (C) Arkadia specifically interacts with SUMO-modified PML-RARα through its SIMs. HEK293 cells were transfected with Flag-Ark-wt or Flag-Ark-SIM123* together with PML-RARα. Cells were treated or not with arsenic for 1 h. Flag immunoprecipates were analyzed by Western blotting using anti-Flag and anti-PML antibodies. The corresponding whole-cell lysates (input) were analyzed using anti-PML antibodies.

Article Snippet: GFP-PML-IV-wt cloned into the pBabe-puro lentiviral vector (Cell Biolabs) was used for infection.

Techniques: Modification, Transfection, Construct, Western Blot, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Arkadia, a Novel SUMO-Targeted Ubiquitin Ligase Involved in PML Degradation

doi: 10.1128/MCB.01019-12

Figure Lengend Snippet: 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).

Article Snippet: GFP-PML-IV-wt cloned into the pBabe-puro lentiviral vector (Cell Biolabs) was used for infection.

Techniques: Transfection, Construct, Western Blot, Staining, Immunostaining