Figure 1.
(A) Immunofluorescence screen. U2OS cells were transfected with a ΔE expressing vector together with each pool of shRNAs targeting the human DUBs. Notch products were detected with anti-myc antibody, or with V1744, to specifically detect NIC (secondary antibodies were coupled with Alexa-488 and Alexa-555, respectively). Nuclei were stained with Hoechst. (B) Western blot analysis of endogenous eIF3f and transfected HA-tagged eIF3f in the presence of the pooled (P1 and P2) or the individual shRNA vectors (#1 to #4 from P1, #5 and #6 from P2) targeting eIF3f. α-tubulin loading control is shown. Under the lanes is shown the quantification of the eIF3f bands (overexpressed or endogenous as indicated), performed with the Quantity One program (Biorad). (C) Quantification of the effect of two shRNA vectors targeting eIF3f (#3 and #4, respectively, in blue and red) on the extranuclear localization of NIC. Transfection and immunofluorescence were performed as in (A). 400 Notch-positive cells were counted for each point and the percentage of cells showing extranuclear V1744 labeling is shown. Error bars represent the SEM of duplicate experiments.
Figure 2.
eIF3f is involved in the deubiquitination of activated Notch.
HEK293T cells were transfected with: (A) ΔE, His-ubiquitin, two increasing doses of eIF3f shRNA #3 (indicated by + and +, respectively), and meIF3f as indicated; (B) ΔE, ΔE-LLFF, NIC or Notch full length (FL), His-ubiquitin, the larger dose of eIF3f shRNA used in (A) (+), and meIF3f as indicated; (C) ΔE, His-ubiquitin, shRNA #3, and two doses of meIF3f WT or Mut as indicated by a filled triangle. In all panels, cells were lysed under denaturing conditions and subjected to Nickel purification. Ni-purified material or WCE (5% of the total lysates) were resolved on SDS-PAGE and analyzed by Western blot using the antibodies indicated on the right of the panels. Notch p120 designates the membrane-anchored form of non-activated full-length Notch, resulting from furin cleavage. Overexpressed meIF3f has a higher apparent molecular weight than the endogenous human eIF3f because of its HA tags. White lines indicate that intervening lanes have been spliced out.
Figure 3.
(A) eIF3f exhibits a DUB activity in a bacterial Ub-GFP assay. Bl21 bacteria were transformed with Ub-GFP-S-Tag plasmid and either GST-fusions or His-fusions as indicated. After protein expression induction, they were lysed by sonication. WCE were analyzed by Western blot using the antibodies indicated under each panel to visualize GFP release from Ub-GFP (WB S-Tag) and to control protein expression (WB GST and His). The GFP product resulting from the DUB activity and the Ub-GFP substrate are indicated by the arrows. (B) eIF3f binds ubiquitin in an eukaryotic Ub-VS assay. HeLa cells were transfected with expression vectors encoding BPLF1 WT or CM (Lanes A–D, J, K), or meIF3f WT or Mut (Lanes E–H, M, N) as indicated. 36 h after transfection, 10 µg of WCE were incubated (or not) with Ub-VS probe (Lanes A to H). Samples were finally analyzed by Western blot as indicated under the panels to monitor Ub-VS binding (Lanes A to H) and to check protein expression (Lanes I to N). β-Actin was used as a loading control. The apparent molecular weights of BPLF1 and meIF3f are 32 kDa and 50 kDa, respectively.
Figure 4.
Colocalization of eIF3f, DTX, and ΔE.
U2OS cells were transfected with expression vectors as indicated on the left. eIF3f, DTX, and ΔE were detected using mouse anti-HA, rabbit anti-VSV, and goat anti-myc, respectively (with CY3-coupled, Alexa 488-coupled, and Alexa 647-coupled secondary antibodies, respectively). Insets represent enlargements (4-fold) of the boxed region, arrows indicate colocalizations, and left panels are merges of the three adjacent panels.
Figure 5.
Interactions of eIF3f and DTX.
(A) Schematic representation of murine eIF3f deletion mutants. The conserved MPN domain is depicted in blue; amino acid coordinates are indicated. (B) HEK293T cells were cotransfected with vectors encoding HA-tagged eIF3f WT or (1–192), and VSV-tagged DTX. Whole cell extracts (WCE, 5% of the lysates) were either directly blotted with antibodies indicated on the right of the lanes or immunoprecipitated first with anti-HA antibody. β-tubulin was used as a loading control. (C) HEK293T cells were cotransfected with vectors encoding Flag-tagged eIF3f (188–361) or (91–361), and VSV-tagged DTX. WCE (5%) were either directly blotted with antibodies indicated on the right of the lanes or immunoprecipitated first with anti-Flag antibody. β-tubulin was used as a loading control. (D) HEK293T cells were cotransfected with vectors encoding the various eIF3f mutants and VSV-DTX. WCE were immunoprecipitated with anti-VSV antibody, and the precipitates were eluted with VSV peptide before being loaded on SDS gels. The eluted material and the WCE (5% of the lysates) were analyzed by Western blotting with antibodies indicated on the right of the lanes. α-tubulin was used as a loading control. (E) Stable cell lines derived form MEFs by retroviral transduction of VSV-DTX or S-tagged eIF3f (either WT or active site mutant) were lysed and subjected to parallel immunoprecipitations with VSV and S-tag antibodies as indicated. The VSV- or Laemmli-eluted material (for VSV and S-tag IPs, respectively) and the WCE (5% of the lysates) were analyzed by Western blotting with antibodies indicated on the right of the lanes. α-tubulin was used as a loading control. White lines indicate that intervening lanes have been spliced out.
Figure 6.
Interaction of eIF3f and activated Notch in the presence of DTX.
HEK293T cells were cotransfected with vectors encoding HA-tagged forms of eIF3f (either WT or Mut in Panel A), VSV-DTX, and myc-tagged Notch constructs (ΔE, ΔE-LLFF, NIC, or FL in Panel B) as indicated above the lanes. Immunoprecipitations were performed with anti-HA antibody. In both panels, immunoprecipitates and WCE (10% of the lysates) were analyzed by Western blot as indicated. α- or β-tubulin were used as loading controls.
Figure 7.
Modulation of the Notch reporter activity (CSL-luciferase) in Notch1-expressing cells cocultured with Dll1-expressing cells (Lanes B–N) or control cells (Lane A). The Notch-expressing cells were first transfected with both CSL-firefly luciferase (Notch reporter) and TK-renilla luciferase (internal control) 24 h before coculture. The CSL-reporter activation corresponds to the ratio between Firefly and Renilla luciferase activities. The relative luciferase activity in the presence of Dll1-expressing cells (Lane B) was defined as 100%. Error bars represent the SEM of triplicate experiments. (A) Effects of increasing doses of expression vectors encoding eIF3f WT (Lanes C–F), Mut (G–J), or (188–361) (K–N) in reporter cells are shown as indicated. Expression levels of eIF3f, Notch, and Dll1 were confirmed by Western blot analysis, using β-tubulin as a loading control. White lines indicate that intervening lanes have been spliced out. (B) shRNAs targeting eIF3f decrease Notch signaling. Increasing doses of eIF3f shRNA P2 (C–E), shRNA #1 (F–H), or a control pool targeting AMSH (I–K) were transfected into the Notch-expressing cells as above. Bottom shows the Western blot analysis of a representative experiment.
Table 1.
List of DUBs and selected oligonucleotide sequences present in the shDUB library Part 2.