Fig 1.
Ikaros is sumoylated in T cells.
(A) 5x105 DN3, DN4 or DP cells were lysed in 1x sample buffer, subjected to SDS-PAGE (8%), and probed with an anti-Ikaros antibody. Arrowheads point to the modified fractions. (B) Cytosolic (C), nuclear (N) or total (T) fractions from 106 EtOH- or 4OHT-treated ILC87-IK1-ER cells were subjected to SDS-PAGE (10%) and analyzed with an anti-Ikaros antibody. (C) Western blot analysis (6% SDS-PAGE) with an anti-Ikaros antibody of total cell extracts from 2x106 4OHT-treated ILC87-IK1-ER cells that were lysed in the presence, absence or combination of N-ethylmaleimide (N), iodacetic acid (IAA, I) or 20 mM DTT, as indicated. (D) 4OHT-treated ILC87-Ik1-ER cells were lysed in buffer containing 1% NP-40 in the presence of NEM and IAA. Nuclear extracts from 50x106 cells were immunoprecipitated with an anti-ER antibody and analyzed on a NuPAGE Novex 3–8% Tris-Acetate gel with the indicated antibodies. In (B), (C) and (D), arrowheads point to unmodified Ik1-ER proteins.
Fig 2.
Impact of Ikaros mutations on sumoylation.
(A) Schematic representation of Ik1-ER deletion mutants. (B) Modification pattern of Ik1-ER deletion mutants. ILC87 cells expressing Ik1-ER or deletion mutants were treated and lysed as described in Fig 1D. 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. (C) 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.
Fig 3.
Sumoylation limits the growth-inhibitory effects of Ikaros.
(A) 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) 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. (C) RT-qPCR analysis of repression of the Mpzl2 and Scn4b 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). (D-F) Competitive growth inhibition assay. (D) 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. (E) 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. (F) 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.
Fig 4.
Sumoylation of Ikaros in B-ALL cells.
(A) 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) 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.