Extended Data Fig. 5: Wee1 is a CRBN neosubstrate targeted by DEG-47.
From: Identification of an allosteric site on the E3 ligase adapter cereblon
(a) Western blot of Wee1, PLK1, and pCDK1 after treatment of MOLM-13 cells with increasing concentration of DEG-47 in the presence or absence of SB-405483 for 24 h. (b) Quantification of n = 3 biologically independent Western blots measuring Wee1 levels shown in a. (c) Western blot of Wee1 levels after treatment with DEG-47 over various time points. (d) Quantification of n = 3 biologically independent Western blots measuring pCDK1 levels shown in a. (e) Quantification of n = 3 biologically independent Western blots measuring PLK1 levels shown in a. (f) Western blot analysis of MOLM-13 cells pretreated with DMSO, MG132, or MLN4924 for 1 h, and then treated with DEG-47 in the presence or absence of SB-405483 for 24 h. (g) Western blot analysis of MOLM-13 CRBN KO or MOLM-13 WT cells treated with DEG-47 in the presence or absence of SB-405483 for 24 h. (h) Western blot analysis of MOLM-13 cells pretreated with pomalidomide or DMSO for 1 h prior to treatment with DEG-47 in the presence or absence of SB-405483 for 24 h. (i) Wee1-HiBiT luminescence signal in cells transiently expressing Wee1-HiBiT or Wee1-HiBiT G322N in HEK293T cells dosed with DEG-47 in the presence or absence of 10 µM SB-405483 for 24 h. (j) Protein expression levels of Wee1 after treatment with indicated compounds in MOLM-13 cells for 24 h. Data are represented as mean ± SD. Comparisons were performed using a one-way ANOVA with Šídák’s multiple comparisons test with * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001. All Western blot, HiBiT, and protein expression data are representative of n = 3 biologically independent samples. For uncropped Western blot images, see Supplementary Fig. 5.
