Fig. 4

SUMO moieties are found at Notch target genes and are bound non-covalently by RBPJ. a ChIP qPCR analysis of SUMO2/3 enrichment at regulatory elements of Notch target genes in HEK293 L3MBTL2 KO or b E2F6 KO cells. Gene Desert served as a negative control (CTRL). The mean of at least three independent biological replicates ± SD. c GST-SUMO1, GST-SUMO2 and GST-SUMO3 fusion proteins were expressed in bacteria and purified. HEK293T cells were transiently transfected with Flag-RBPJ and whole cell extract was incubated with GST fusion proteins immobilized on sepharose beads. Flag-RBPJ binds non-covalently to GST-SUMO2 and GST-SUMO3 but not to GST-only. (d, upper) Schematic representation of the wild type and the mutated SIM of RBPJ. (d, lower) GST-SUMO2 fusion protein was expressed in bacteria and purified. HEK293T cells were transiently transfected with GFP-RBPJ wild type or GFP-RBPJ IV/AA mutant and whole cell extracts were incubated with GST fusion protein immobilized on sepharose beads. (e, upper) Electrophoretic Mobility Shift Assay (EMSA) analysis of RBPJ wt and RBPJ IV/AA mutant binding to DNA. Oligomeric duplex DNA probe with RBPJ binding sites (bold): 5'-CCT GGA ACT ATT TTC CCA CGG TGC CCT TCC GCC CAT TTT CCC ACG AGT CG -3'. DNA–protein complexes are indicated as A and B. Supershifted complexes after addition of Flag antibodies are indicated by a and b. The asterisk indicates a nonspecific background band. (e, lower) Western blot showing the in vitro translated Flag-RBPJ proteins used in the EMSA. (f, left) Transactivation capacities of RBPJ-VP16 fusion proteins. Hela cells were cotransfected with either RBPJ-VP16 wt or RBPJ-VP16 IV/AA mutant together with 12 × CSL-RE-Luc reporter construct containing 12 RBPJ DNA binding sites upstream of the luciferase gene. The mean of at least four independent biological replicates ± SD is shown (***p < 0.0001, unpaired students T-test). (f, right) Western blot show slightly reduced expression of the RBPJ-VP16 (IV/AA) mutant. HeLa cells were transiently transfected with RBPJ-VP16 (wt) or RBPJ-VP16 (IV/AA) mutant. 24 h after transfection cells, where lysed and expression of the VP16 were analysed by western blotting. Actin expression served as a loading control