Fig. 2: CAND2 affects the cellular landscape of SCFs and the degradation of SCF target proteins.

A CAND2-KO altered the assembly levels of multiple SCFs in HEK293 cells. WT and CAND2-KO cells expressing endogenous ^3xFLAGCUL1 were incubated with an anti-FLAG antibody in the presence of the CUL1^split·RBX1 “sponge” protein, MLN4924, and quinoline-8-thiol (8TQ). Relative levels of F-box proteins (FBPs) assembled with ^3xFLAGCUL1 were quantified using mass spectrometry, and FBPs with statistically significant changes are shown. Data are presented as mean ± SEM; n = 3 biological replicates. Unpaired two-tailed Student’s t-test. B, C CAND2-KO slows the degradation of IRP2 in JMSU-1 cancer cells. B Immunoblotting of IRP2, CAND2, and GAPDH at the indicated time points following the start of FAC-induced degradation of IRP2 in the indicated JMSU-1 cells. C Quantitative analyses of IRP2 degradation in (B). Data are presented as mean ± SEM; n = 3 independent experiments. D, E Effects of CAND2-KO on global protein turnover in JMSU-1 cells. D Schematics illustrating the experimental design. Cell samples were prepared in duplicate. E Summary of log₂ fold change (FC) of protein half-lives resulting from CAND2-KO in JMSU-1 cells. The dashed lines mark the log₂(FC) cutoffs of 0.5 and −0.5. A few previously characterized SCF target proteins detected in this assay are listed in the table on the right. F Top Gene Ontology (GO) categories associated with the proteins colored red and green in (E). G, H CAND2-KO slows the degradation of BCL10 in JMSU-1 cells. G Immunoblotting of BCL10 and GAPDH at the indicated time points after the addition of cycloheximide (CHX) in the indicated JMSU-1 cells. H Quantitative analyses of BCL10 degradation in (G). Data are presented as mean ± SEM; n = 3 independent experiments. Source data are provided as a Source Data file.