Journal: Nature Communications

Article Title: CRL4 DCAF12 regulation of MCMBP ensures optimal licensing of DNA replication

doi: 10.1038/s41467-025-64258-5

Figure Lengend Snippet: a , d QIBC plots of MCM4-Halo cells treated with siRNAs as indicated. Lines denote medians; n = 4973 (minimum cells) ( a ) or 5003 (minimum cells) ( d ) per condition. b , e Quantification of QIBC plots; each bar indicates the median of mean intensity (data are mean ± s.d.; n = 4 (2 biological replicates, each with 2 technical replicates) for both ( b , e ). c , f Representative images; the pseudo-color gradient indicates the mean fluorescent intensity (MFI). Scale bar, 20 µm. g Schematic representation of the tandem affinity purification strategy. h , i CRL4 DCAF12 -associated proteins identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Non-related ubiquitin ligases CRL4 DCAF4 in ( h ) or SCF FBXL6 in ( i ) were used as a control. Proteins that were significantly enriched with CRL4 DCAF12 compared to controls are in the upper right quadrants ( n = 2 biological replicates). j Affinity purification (AP) of a panel of CRLs. HEK293T cells were transfected with an empty vector (EV) or StrepII-FLAG-tagged CRL constructs for 48 h and treated with MLN4924 for 6 h before harvesting. Whole-cell lysates were subjected to AP with Strep-TactinXT resin. M denotes a molecular marker. k Affinity purification of N-terminal StrepII-FLAG-tagged DCAF12 (SF-DCAF12) and immunopurification of HA-tagged MCMBP, MOV10, and C-terminal degron-lacking mutants. HEK293T cells were co-transfected with SF-DCAF12 and either an empty vector or specified gene constructs. MLN4924 was added for the final 6 h before harvest. Whole-cell lysates were subjected to affinity purification (AP) with Strep-TactinXT resin or anti-HA immunoprecipitation (IP). The left panel shows 1% input samples, the upper right panel shows Strep-Tactin AP, and the lower right panel shows anti-HA IP. j , k are representatives of three independent replicates with similar results. For ( h , i ), statistical analysis was performed in Perseus (version 1.6.15.0) using a two-sample Student’s t test (two-sided; S ₀ = 0.1) with permutation-based false discovery rate (FDR) correction (FDR = 0.05). P values were calculated by ordinary one-way ANOVA with Dunnett’s test ( b , e ); n.s. (not significant) indicates p > 0.1. (A.U. Arbitrary Units). Source data are provided as a file.

Article Snippet: The human kidney cancer cell line HEK293T (ATCC, CRL-1573), colon cancer cell line HCT116 (ATTC, CCL-247), and its derivatives were maintained in Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with 10% FBS, and penicillin (BB Pharma), streptomycin (Merck), and gentamycin (Sandoz), and cultured in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: Affinity Purification, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Ubiquitin Proteomics, Control, Transfection, Plasmid Preparation, Construct, Marker, Immu-Puri, Immunoprecipitation

Journal: Nature Communications

Article Title: CRL4 DCAF12 regulation of MCMBP ensures optimal licensing of DNA replication

doi: 10.1038/s41467-025-64258-5

Figure Lengend Snippet: a A schematic describing the experimental approach for understanding the role of CUL4-DCAF12 in MCM2-7 assembly (see text for details). b Halo-immunoprecipitation (Halo-IP) of whole cell lysates. Cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. c Quantification of Halo-IP in ( b ); IP products in control cells have been considered as 100% (data are mean ± s.d.; n = 2 biological replicates). d Halo-IP of nuclear fraction. Cells were treated with siDCAF12 as indicated. IPs in ( b , d ) were done in low salt condition (150 mM NaCl). e Quantification of Halo-IP in ( d ); IP products in control cells have been considered as 100% (data mean ± s.d.; n = 2 biological replicates). f Sum fluorescence intensity (SFI) of PLA foci for the MCMBP-MCM2 antibody pair in U2OS cells treated with control or siRNA against DCAF12 . Lines denote medians; n = 3454 (minimum cells per condition). g Representative images from ( f ). This experiment was independently repeated two times with similar results. Scale bar, 10 µm. h SFI of PLA foci for the CDT1-MCM2 antibody pair in G1/S phase of PLA-positive U2OS cells treated with control or siRNA against DCAF12. Lines denote medians; n = 746 cells per condition. i Representative images from ( h ). This experiment was independently repeated two times with similar results. Scale bar, 10 µm. j Heatmap, derived from mass spectrometry analysis of HEK293T cells co-transfected with StrepII-FLAG-tagged DCAF12, shows log2 intensities of indicated proteins. Cells were treated with either DMSO (CTRL) or 10 µM MG132 for 6 h prior to protein purification. k Schematic representation of human DCAF12 and its N-terminally truncated mutants. Two predicted nuclear localization signals (NLS) and H-box are highlighted. l , m QIBC plots of the cytoplasm of HCT116 cells stained for StrepII-FLAG-tagged DCAF12 (SF-DCAF12) ( l ) or MCMBP ( m ) protein after indicated DCAF12 constructs were inducibly expressed. Lines denote medians; n = 1051 (minimum cells) ( l ) or 1105 (minimum cells) ( m ) per condition. P values in ( f , h ) were calculated by two-tailed unpaired t -test. Source data are provided as a file.

Article Snippet: The human kidney cancer cell line HEK293T (ATCC, CRL-1573), colon cancer cell line HCT116 (ATTC, CCL-247), and its derivatives were maintained in Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with 10% FBS, and penicillin (BB Pharma), streptomycin (Merck), and gentamycin (Sandoz), and cultured in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: Immunoprecipitation, Control, Fluorescence, Derivative Assay, Mass Spectrometry, Transfection, Protein Purification, Staining, Construct, Two Tailed Test