Additional File 2: Figures S1-S4
Additional figures:
Figure S1. (a) HEK 293T cells were transiently transfected with GFP-RBPJ plasmid. Cell lysates were subjected to GFP immunoprecipitation. Control cells were transfected with pcDNA GFP plasmid. (b) GST-RBPJ fusion protein was expressed in bacteria and purified. Fragments of L3MBTL2 were labeled with [35S] methionine, in vitro translated in RRL system and incubated with GST RBPJ fusion protein
immobilized on sepharose beads. (c) GST-L3MBTL2 fusion protein was expressed in bacteria and purified. Fragments of RBPJ were radioactively labeled with [35S] methionine, in vitro translated in RRL system and incubated with GST-L3MBTL2 fusion protein immobilized on sepharose beads.
Figure S2. (a) Schematic representation of the targeting strategy for generating CRISPR/Cas9 mediated RBPJ depletion in HEK 293 cells (Exon: ENSE00003633263). (b) Western Blot analysis of endogenous RBPJ in wild type HEK293 and in RBPJ-depleted cells (clones A2 and A12). GAPDH served as a loading control. (c) mRNA level of RBPJ in CRISPR/Cas9 mediated RBPJ depletion in HEK
293 (clone A12). Data was normalised to TBP, GAPDH served as a positive control. . (*P < 0.05, **P < 0.01, ***P < 0.001, [NS] not significant, unpaired Student's t-test). The mean of at least three independent biological replicates ±SD is shown. (d) Chromatin Immunoprecipitation of endogenous RBPJ and its binding at regulatory elements of Notch target genes in wild type and in RBPJ depleted cells (clone A12). Gene Desert served as a negative control (CTRL). The mean of at least three independent biological replicates ±SD.
Figure S3. (a) ChIP qPCR analysis of SUMO2/3 enrichment at regulatory elements of Notch target genes in HEK293 cells. (b) GST-SUMO2 fusion protein was expressed in bacteria and purified. HEK293T cells were transiently transfected with GFP-RBPJ wild type or GFP-RBPJ ΔNTD mutant and whole cell extracts were incubated with GST fusion protein immobilized on sepharose beads. (c, upper) Subcellular localisation of GFP-RBPJ wt and GFP-RBPJ IV/AA mutant. Hela cells were transiently transfected with GFP-RBPJ wt or GFP-RBPJ IV/AA mutant and fixed 24h after transfection. (c,
lower panel) Western blot show slightly reduced expression of the GFP-RBPJ (IV/AA) mutant. HeLa cells were transiently transfected with GFP-RBPJ expression vectors. 24 hours after transfection cells where lysed and expression of the GFP-fusions were analysed by western blotting. Actin expression served as a loading control. (d, upper) Transactivation capacities of RBPJ (wt) and RBPJ (IV/AA) mutant together with NICD. HelaRBPJ-KO cells were cotransfected with NICD together with either Flag-RBPJ-wt or Flag-RBPJ IV/AA mutant and the 12 x 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 (ns, p ≥ 0.05; ***, p<0.0001, unpaired students T-test). Western blot show slightly reduced expression of the Flag-RBPJ (IV/AA) mutant. HeLa cells were transiently transfected with GFP-RBPJ expression vectors. 24 hours after transfection cells where lysed and expression of Flag-tagged proteins were analysed by western blotting. Actin expression served as a loading control.
Figure S4 (a) HEK 293T cells were co-transfected with Flag L3MBTL2 and GFP RBPJ. The cells were treated for 24h with 10μM ML-792 or the vehicle. Cell lysates after extraction were subjected for GFP immunoprecipitation. Control cells were transfected with pcDNA GFP plasmid. (b) HEK 293T cells were co-transfected with Flag-RBPJ and GFP-NICD1. The cells were treated for 24h with 10μM ML-792 or DMSO. Cell lysates after extraction were subjected for GFP immunoprecipitation. 851 Control cells were transfected with pcDNA GFP plasmid.