Sumoylation of Ikaros in B-ALL cells.

<p><b>(A)</b> Detection of Ikaros proteins by western blot in whole cell extracts from human peripheral blood mononuclear cells (PBMC), primary leukemic cells from a B-ALL patient, and the ACC42, RS4;11 and Tom-1 B-ALL cell lines. <b>(B)</b> Detection of Ikaros sumoylation in whole cell extracts from B-ALL patient #H4524 and the cell line ACC42. Protein extracts were immunoprecipitated with an anti-Ikaros antibody, and probed with an anti-Ikaros antibody, or with a mix of anti-SUMO1 and anti-SUMO2/3 antibodies. Open arrowheads point to the Ik1 and Ik2 isoforms; asterisks to IgGs. Note that a sumoylated protein comigrates with Ik1, presumably corresponding to sumoylated Ik2.</p

Impact of Ikaros mutations on sumoylation.

<p><b>(A)</b> Schematic representation of Ik1-ER deletion mutants. (<b>B</b>) Modification pattern of Ik1-ER deletion mutants. ILC87 cells expressing Ik1-ER or deletion mutants were treated and lysed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157767#pone.0157767.g001" target="_blank">Fig 1D</a>. Nuclear extracts were immunoprecipitated with an anti-ER antibody, separated by SDS-PAGE (6%) and analyzed with anti-Ikaros antibodies against C-terminal or N-terminal epitopes. (<b>C</b>) Nuclear extracts from 50x10⁶ ILC87 cells expressing Ik1-ER or the indicated point mutants were immunoprecipitated with an anti-ER antibody, separated on a NuPAGE Novex 3–8% Tris-Acetate gel and analyzed with an anti-Ikaros antibody. The pattern of the various modified proteins is schematized on the right and the corresponding modifications indicated. In (B) and (C), arrowheads point to unmodified proteins.</p

Sumoylation limits the growth-inhibitory effects of Ikaros.

<p><b>(A)</b> Western blot showing similar level of Ik1-ER and Ik-TM-ER proteins in nuclear extracts of ILC87-Ik1-ER and ILC87-Ik-TM-ER cells treated with EtOH or 4OHT. <b>(B)</b> Scatter plots showing the distributions of the fold changes (4OHT- vs EtOH-treated cells, expressed as log2 values) in the 2 independent analyses performed with ILC87-Ik1-ER cells (top panel), or between representative analyses performed with ILC87-Ik1-ER and ILC87-Ik-TM-ER cells (bottom panel). The red diagonal highlights the theoritical position for probe sets with similar fold-changes. <b>(C)</b> RT-qPCR analysis of repression of the <i>Mpzl2</i> and <i>Scn4b</i> genes in ILC87-Ik1-ER and ILC87-Ik-TM-ER cells treated with 4OHT for 24h, in 2 independent experiments (duplicate measurements in each case). <b>(D-F)</b> Competitive growth inhibition assay. <b>(D)</b> Experimental setup: ILC87 cells transduced with IK1-ER or Ik-TM-ER (GFP⁺) were mixed at a 1:1 ratio with ILC87 cells (or ILC87 cells mock-transduced with an empty Mig-NGFR retrovirus) and cultured for 6 days in the presence of EtOH or 4OHT. <b>(E)</b> Proportion of GFP⁺ cells in living cells of EtOH and 4OHT-treated ILC87-Ik1-ER and ILC87-Ik-TM-ER cells in a representative experiment. <b>(F)</b> Growth inhibition over time by Ik1-ER and Ik-TM-ER (ratio of GFP⁺ cells in 4OHT-treated over EtOH-treated samples; average of 4 experiments). Statistical significance was evaluated with a Student's t-test.</p