Fig 2: CAND2 promotes the exchange of F-box proteins (FBPs) but less efficiently than CAND1 does.A, B CAND2 promotes the assembly of new SCF complexes by accelerating the disassembly of pre-existing SCFs. A Schematic diagram of the experimental design. DKO cells expressing endogenous 3xFLAGCUL1 were subjected to IP with an anti-FLAG antibody in the presence of excess SKP1Sortase·SKP2ΔLRR chase protein and equal concentrations of recombinant CAND1 or CAND2. Samples were immunoblotted with the indicated antibodies (B). Representative results from three independent experiments are shown. C SILAC-based quantitative proteomic assay assessing FBP exchange levels in cell lysates. The experimental procedure is illustrated (upper flowchart), and general profiles of FBP exchange under different conditions are shown (bottom graph). Black bars: median exchange. D Exchange levels of CSN and the common subunits of SCF. Results from three biological replicates are shown. H/L ratio: heavy/light ratio. E Exchange levels of FBPs detected in all four experimental groups. Results from three biological replicates are shown. F Exchange levels of FBPs that displayed increased exchange only in response to CAND1. Data are presented as mean ± SEM; n = 3 biological replicates except for FBXO5 in the 1x CAND1 group (n = 2 biological replicates). G Exchange levels of FBPs detected only in the absence of CAND1. Results from three biological replicates are shown. Source data are provided as a Source Data file.

Fig 3: CAND2 promotes SCF-mediated protein degradation.A CAND2 binds to unneddylated CUL1 in human cell lysates. Whole-cell lysates (WCL) from HEK293 cells of the indicated genotypes were immunoprecipitated (IP) with an anti-HA antibody. Cells were treated with 1 μM MLN4924 for 1 h before harvesting. Samples were immunoblotted with anti-CUL1, anti-HA, and anti-GAPDH antibodies. DKO: CAND1 and CAND2 double knockout cells; DKOCAND1-HA: DKO cells expressing transgenic CAND1HA; DKOCAND2-HA: DKO cells expressing transgenic CAND2HA. Representative results from three independent experiments are shown. B, C CAND2 rescues the TNFα-induced degradation of p-IκBα in DKO cells. B Immunoblotting of HA (CAND1 or CAND2), IκBα, and GAPDH at the indicated time points after the addition of TNFα to the indicated cells in the presence of cycloheximide (CHX). C Quantification of the relative p-IκBα levels in (B). Data are presented as mean ± SEM; n = 3 independent experiments. D, E Both CAND1 and CAND2 are required for proper degradation of IRP2 in HEK293 cells. D Immunoblotting of IRP2, FBXL5, and GAPDH at the indicated time points after the addition of ferric ammonium citrate (FAC) to induce the accumulation of FBXL5 and the degradation of IRP2 in the indicated cells. KO: knockout. E Quantitative analyses of IRP2 degradation in (D). Data are presented as mean ± SEM; n = 3 independent experiments except for DKO (n = 4). F–H CAND2-KO impaired the enrichment of the IRP2-bound active SCF complex. F Schematic diagram of the experimental procedure. G Samples from cells of the indicated genotypes were prepared as described in (F) and immunoblotted with anti-HA (FBXL5), anti-FLAG (IRP2), and anti-CUL1 antibodies. The normalized signal intensity ratios of co-immunoprecipitated neddylated CUL1 (CUL1N8) and FLAGIRP2 are shown below the blots. H Quantitation of results in (G). Data are presented as means of two independent experiments distinguished by dots with different shapes and colors. Source data are provided as a Source Data file.

Fig 4: 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 CUL1split·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.

Fig 5: FBP changes the koff but not the kon of CUL1·CAND2.A Binding (kon) of CAND1 or CAND2 to CUL1 preassembled with SKP1·SKP2. kobs of CFPCAND1·CUL1FlAsH or CFPCAND2·CUL1FlAsH association when 5 nM CFPCAND1 or CFPCAND2 was mixed with increasing concentrations of SKP1·SKP2-bound CUL1FlAsH (Supplementary Fig. 7). The linear slope gave the kon. Data are presented as mean ± SEM; n = 3 independent experiments. B–D SKP1·SKP2 increases the koff of CUL1·CAND1 and CUL1·CAND2 to similar degrees. Real-time detection of CFP fluorescence in a stopped-flow apparatus upon the addition of 80 nM SKP1·SKP2 to 5 nM CFPCAND1·CUL1FlAsH (B) or CFPCAND2·CUL1FlAsH (C). D kobs of CFPCAND1·CUL1FlAsH or CFPCAND2·CUL1FlAsH dissociation when increasing concentrations of SKP1·SKP2 were mixed with 5 nM CFPCAND1·CUL1FlAsH or CFPCAND2·CUL1FlAsH (Supplementary Fig. 8). Fitting with hyperbola gave the koff and KM. Data are presented as mean ± SEM; n = 5 independent experiments. Source data are provided as a Source Data file.