Figure 4.
NOTCH2 binds KLHL6 in a degron that is different from FBXW7-degron. (A, left) Schematic representation of WT or mutated NOTCH2 interacting (+) or not (−) with KLHL6 is shown. (A, right) Immunoblot analysis for the indicated proteins from the WCL or FLAG-IP samples from HEK293T cells stably expressing hemagglutinin (HA)-tagged KLHL6 and overexpressing the indicated FLAG-tagged WT or mutated NOTCH2. (B) Same as that in panel A, but the indicated NOTCH2 mutants are used. (C, top) Same as that in panel A, but the indicated NOTCH2 mutants are used. (C, bottom) Alignment of the NOTCH2 degron in different species. The conserved Ser-Pro-His degrons are shown in red. (D, left) Schematic representation of NOTCH2 (WT), NOTCH2 (S2156A), and DLBCL-associated NOTCH2 mutants, NOTCH2 (R2400∗) and NOTCH2 (Q2140∗). (D, right) Immunoblot analysis for the indicated proteins from the WCL in U2932 cells stably expressing HA-tagged NOTCH2 (WT), NOTCH2 (S2156A) or DLBCL-associated NOTCH2 mutants, NOTCH2 (R2400∗) and NOTCH2 (Q2140∗). (E, left) Flow cytometry analysis for the plasma membrane NOTCH2 in U2932 cells stably expressing HA-tagged NOTCH2 (WT), NOTCH2 (S2156A) or DLBCL-associated NOTCH2 mutants, NOTCH2 (R2400∗) and NOTCH2 (Q2140∗). (E, right) Quantification of the MFI of the plasma membrane NOTCH2 in U2932 cells (mean ± SD; n = 3 independent experiments; one-way ANOVA; ∗∗∗∗P ≤ .0001). Unless otherwise noted, immunoblots are representative of 3 independent experiments. ANK, ankyrin; RAM, RBP-J associated module; TAD, transactivation domain.

NOTCH2 binds KLHL6 in a degron that is different from FBXW7-degron. (A, left) Schematic representation of WT or mutated NOTCH2 interacting (+) or not (−) with KLHL6 is shown. (A, right) Immunoblot analysis for the indicated proteins from the WCL or FLAG-IP samples from HEK293T cells stably expressing hemagglutinin (HA)-tagged KLHL6 and overexpressing the indicated FLAG-tagged WT or mutated NOTCH2. (B) Same as that in panel A, but the indicated NOTCH2 mutants are used. (C, top) Same as that in panel A, but the indicated NOTCH2 mutants are used. (C, bottom) Alignment of the NOTCH2 degron in different species. The conserved Ser-Pro-His degrons are shown in red. (D, left) Schematic representation of NOTCH2 (WT), NOTCH2 (S2156A), and DLBCL-associated NOTCH2 mutants, NOTCH2 (R2400∗) and NOTCH2 (Q2140∗). (D, right) Immunoblot analysis for the indicated proteins from the WCL in U2932 cells stably expressing HA-tagged NOTCH2 (WT), NOTCH2 (S2156A) or DLBCL-associated NOTCH2 mutants, NOTCH2 (R2400∗) and NOTCH2 (Q2140∗). (E, left) Flow cytometry analysis for the plasma membrane NOTCH2 in U2932 cells stably expressing HA-tagged NOTCH2 (WT), NOTCH2 (S2156A) or DLBCL-associated NOTCH2 mutants, NOTCH2 (R2400∗) and NOTCH2 (Q2140∗). (E, right) Quantification of the MFI of the plasma membrane NOTCH2 in U2932 cells (mean ± SD; n = 3 independent experiments; one-way ANOVA; ∗∗∗∗P ≤ .0001). Unless otherwise noted, immunoblots are representative of 3 independent experiments. ANK, ankyrin; RAM, RBP-J associated module; TAD, transactivation domain.

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