Figure 3. NOTCH2 mutations in HCS impair the FBW7 recognizable phospho-degron motif.

A. Domain structure of human NOTCH2 protein, positions of mutations reported in patients with HCS, and truncation mutations used in this study (namely NOTCH2 ICD Δ1 and Δ2).

B. FBW7 consensus degron motifs in human cyclin E, c-Jun, Presenilin, and NOTCH2.

C. IB analysis of WCL and anti-HA or -Flag IPs derived from 293T cells transfected with EV or indicated Flag-NOTCH2 ICD (WT, Δ1, or Δ2). At 24 h post-transfection, cells were treated with 15 μM MG132 for 12 h before harvesting.

D. IB analysis of WCL derived from 293T cells transfected with Flag-NOTCH2 ICD, HA-FBW7, and HA-GSK3β as indicated. GFP served as the internal control for transfection efficiency.

E. IB analysis of WCL and anti-Flag IPs derived from 293T cells transfected with Flag-NOTCH2 ICD (WT, Δ1, or Δ2), Myc-Ub, HA-FBW7, and GSK3β as indicated. At 24 h post-transfection, cells were treated with 15 μM MG132 for 12 h before harvesting.

F. IB analysis of WCL derived from HeLa cells transfected with Flag-NOTCH2 ICD (WT, Δ1, or Δ2), HA-FBW7, and HS-GSK3β. At 48 h post-transfection, cells were treated with 20 μg/mL CHX and harvested at the indicated time points.

G. Alignment of NOTCH2 sequences surrounding the putative FBW7 degron motif from different species.

H. IB analysis of WCL and anti-HA or -Flag IPs derived from 293T cells transfected with EV or Flag-NOTCH2 ICD (WT or T2416A) and HA-FBW7. At 24 h post-transfection, cells were treated with 15 μM MG132 for 12 h before harvesting.

I. In vitro binding of HA-FBW7 with GST-NOTCH2 ICD. Bacterially purified GST or GST-NOTCH2 ICD (WT, Δ2, or T2416A) protein treated with active GSK3 as indicated was incubated with WCL derived from 293T cells transfected with HA-FBW7. The bound HA-FBW7 protein with GST-NOTCH2 ICD was eluted and subjected to IB analysis.

J. IB analysis of WCL derived from 293T cells transfected with Flag-NOTCH2 ICD (WT or T2416A), HA-FBW7, and HA-GSK3β as indicated. GFP served as an internal control for transfection efficiency.

K. IB analysis of WCL derived from HeLa cells transfected with Flag-NOTCH2 ICD (WT or T2416A), HA-FBW7, and HA-GSK3β. At 48 h post-transfection, cells were treated with 20 μg/mL CHX and harvested at the indicated time points.

L. IB analysis of WCL and anti-Flag IPs from 293T cells transfected with Flag-NOTCH2 ICD (WT or T2416A), Myc-Ub, HA-FBW7, and GSK3β as indicated. At 24 h post-transfection, cells were treated with 15 μM MG132 for 12 h before harvesting.

See also Figure S3