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Name: mcmbp
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a, A schematic describing the experimental approach for understanding the role of CUL4-DCAF12 <t>in</t> <t>MCM2-7</t> assembly (see text for details). b, Halo-immunoprecipitation (Halo IP) of whole cell lysates followed by western blotting, cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. This IP was done at low salt concentration (150 mM NaCl) c, Quantification of Halo-IP products based on western blots in b ; IP products upon control siRNA treatments have been considered as 100% (data are mean ± s.d.; n = 2 biological replicates). d, Halo IP of whole cell lysates followed by western blotting, cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. This IP was done at a high salt concentration (500 mM NaCl). e, Quantification of Halo-IP products based on western blots in d ; IP products upon control siRNA treatments have been considered as 100% (data mean ± s.d.; n = 2 biological replicates). e, 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 hours prior to protein purification. g, Schematic representation of human DCAF12 and its N-terminally truncated mutants. Two predicted nuclear localization signals (NLS) and H-box are highlighted. h, i, QIBC plots of the cytoplasm of U2OS cells stained for StrepII-tagged DCAF12 ( h ) or <t>MCMBP</t> ( i ) protein after indicated DCAF12 constructs were inducibly expressed. Lines denote medians; n ≈ 1,000 cells per condition.

Mcmbp, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "CRL4 DCAF12 regulation of MCMBP ensures optimal licensing of DNA replication"

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

Journal: bioRxiv

doi: 10.1101/2024.11.26.625391

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 followed by western blotting, cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. This IP was done at low salt concentration (150 mM NaCl) c, Quantification of Halo-IP products based on western blots in b ; IP products upon control siRNA treatments have been considered as 100% (data are mean ± s.d.; n = 2 biological replicates). d, Halo IP of whole cell lysates followed by western blotting, cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. This IP was done at a high salt concentration (500 mM NaCl). e, Quantification of Halo-IP products based on western blots in d ; IP products upon control siRNA treatments have been considered as 100% (data mean ± s.d.; n = 2 biological replicates). e, 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 hours prior to protein purification. g, Schematic representation of human DCAF12 and its N-terminally truncated mutants. Two predicted nuclear localization signals (NLS) and H-box are highlighted. h, i, QIBC plots of the cytoplasm of U2OS cells stained for StrepII-tagged DCAF12 ( h ) or MCMBP ( i ) protein after indicated DCAF12 constructs were inducibly expressed. Lines denote medians; n ≈ 1,000 cells per condition.

Figure Legend 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 followed by western blotting, cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. This IP was done at low salt concentration (150 mM NaCl) c, Quantification of Halo-IP products based on western blots in b ; IP products upon control siRNA treatments have been considered as 100% (data are mean ± s.d.; n = 2 biological replicates). d, Halo IP of whole cell lysates followed by western blotting, cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. This IP was done at a high salt concentration (500 mM NaCl). e, Quantification of Halo-IP products based on western blots in d ; IP products upon control siRNA treatments have been considered as 100% (data mean ± s.d.; n = 2 biological replicates). e, 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 hours prior to protein purification. g, Schematic representation of human DCAF12 and its N-terminally truncated mutants. Two predicted nuclear localization signals (NLS) and H-box are highlighted. h, i, QIBC plots of the cytoplasm of U2OS cells stained for StrepII-tagged DCAF12 ( h ) or MCMBP ( i ) protein after indicated DCAF12 constructs were inducibly expressed. Lines denote medians; n ≈ 1,000 cells per condition.

Techniques Used: Immunoprecipitation, Western Blot, Concentration Assay, Control, Derivative Assay, Mass Spectrometry, Transfection, Protein Purification, Staining, Construct

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Journal: bioRxiv

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

doi: 10.1101/2024.11.26.625391

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 followed by western blotting, cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. This IP was done at low salt concentration (150 mM NaCl) c, Quantification of Halo-IP products based on western blots in b ; IP products upon control siRNA treatments have been considered as 100% (data are mean ± s.d.; n = 2 biological replicates). d, Halo IP of whole cell lysates followed by western blotting, cells were treated with siRNA against DCAF12 (siDCAF12) as indicated. This IP was done at a high salt concentration (500 mM NaCl). e, Quantification of Halo-IP products based on western blots in d ; IP products upon control siRNA treatments have been considered as 100% (data mean ± s.d.; n = 2 biological replicates). e, 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 hours prior to protein purification. g, Schematic representation of human DCAF12 and its N-terminally truncated mutants. Two predicted nuclear localization signals (NLS) and H-box are highlighted. h, i, QIBC plots of the cytoplasm of U2OS cells stained for StrepII-tagged DCAF12 ( h ) or MCMBP ( i ) protein after indicated DCAF12 constructs were inducibly expressed. Lines denote medians; n ≈ 1,000 cells per condition.

Article Snippet: Primary antibodies used for immunofluorescence (IF) were as follows: CDT1 (rabbit, Abcam, ab202067, 1:2,000), Cyclin D1 (rabbit, Proteintech, 26939-1-AP, 1:1,000), GFP (rabbit, Proteintech, PABG1, 1:5,000), MCMBP (rabbit, Novus Biologicals, NBP1-90746, 1:1,000), MCM2 (rabbit, Proteintech, 10513-1-AP, 1:1,000), MCM3 (mouse, Santa Cruz, sc-390480, 1:1,000), MCM4 (rabbit, Proteintech, 13043-1-AP, 1:1,000), MCM5 (rabbit, Proteintech, 11703-1-AP, 1:1,000), MCM6 (mouse, Novus Biologicals, H00004175-M04, 1:1,000), MCM7 (mouse, Santa Cruz, sc-9966, 1:1,000), PCNA (human, Immuno Concepts, 2037, 1:1,000), RAD51 (rabbit, BioAcademia, 70-012, 1:1,000), Strep II Tag (mouse, Novus Biologicals, NBP2-43735, 1:1,000), γH2AX (Ser139) (rabbit, Abcam, ab81299, 1:1,000).

Techniques: Immunoprecipitation, Western Blot, Concentration Assay, Control, Derivative Assay, Mass Spectrometry, Transfection, Protein Purification, Staining, Construct

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.

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: Primary antibodies used for western blotting were as follows: α-tubulin (mouse, Proteintech, 66031-1-Ig, 1:1,000), β-actin (mouse, Santa Cruz, sc-69879, 1:1,000), CCNA (home-made serum), DDB1 (rabbit, Zymed, 34-2300, 1:1,000), GART (mouse, Santa Cruz, sc-166379, 1:1,000), γH2AX (rabbit, Proteintech, 83307-2-RR, 1:1,000; mouse, Milipore, 05-636, 1:1,000), HA (rabbit, Cell Signaling, 3724, 1:1,000), MCMBP (rabbit, Proteintech, 19573-1-AP, 1:1,000; rabbit, Atlas Antibodies, HPA038481, 1:1,000), MCM2 (rabbit, Proteintech, 10513-1-AP, 1:1,000), MCM2 (rabbit, ABClonal, A1056, 1:1,000), MCM3 (mouse, Santa Cruz, sc-390480, 1:1,000), MCM3 (rabbit, ABClonal, A1060, 1:1,000), MCM4 (rabbit, Proteintech, 13043-1-AP, 1:1,000), MCM5 (rabbit, Proteintech, 11703-1-AP, 1:1,000), MCM6 (rabbit, Proteintech, 13347-2-AP, 1:1,000), MCM7 (mouse, Santa Cruz, sc-9966, rabbit, Proteintech, 11225-1-AP, 1:1,000), p53 (mouse, Santa Cruz, sc-126, 1:1,000), PARP1 (mouse, Proteintech, 66520-1-Ig, 1:1,000), PCNA (mouse, Santa Cruz, sc-56, 1:1,000), pH3 (Ser10) (rabbit, Cell Signaling, 53348, 1:1,000), RPS3A (rabbit, ABClonal, A5885, 1:1,000), RPS6 (mouse, Santa Cruz, sc-74459, 1:1,000), SKP1 (rabbit, Cell Signaling, 12248, 1:1,000).

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

$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.$

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.

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

Journal: bioRxiv

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

doi: 10.1101/2024.11.26.625391

Figure Lengend Snippet: a, QIBC plots of MCM4-Halo cells immunostained for MCMBP after treatment with siRNAs against cullins (as indicated). Lines denote medians; n ≈ 5,000 cells per condition. b, Quantification of QIBC plots in a ; each bar indicates the median of mean intensity (data are mean ± s.d.; n = 3 technical replicates). c, QIBC plots of MCM4-halo cells immunostained for MCMBP after treatment with siRNAs against the DCAF family of adaptors (as indicated). Lines denote medians; n ≈ 5,000 cells per condition. d, Quantification of QIBC plots in c ; each bar indicates the median of mean intensity (data are mean ± s.d.; n = 3 technical replicates). e, Percentage distribution of individual cell cycle phases of MCM4-Halo cells after treatment with control siRNA or siRNAs against CDT1 , AMBRA1 , or DCAF12 . Individual phases were defined according to the DAPI profile based on sub- stratification shown in Extended Data Fig. 1h. f, QIBC plots showing the percentage distribution of MCMBP in indicated cell cycle phases. Lines denote medians; n ≈ 6,800 cells divided into G1, S, and G2 phases. g, qPCR-based analysis of MCMBP mRNA levels after indicated siRNA treatments; normalized with respect to siCTRL treatment as 1 (n = 3 technical replicates). h, Schematic representation of the tandem affinity purification strategy. i-j, CRL4 DCAF12 -associated proteins identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The composition of purified complexes from both steps was analyzed by LC-MS/MS. Non-related ubiquitin ligases ( i ) CRL4 DCAF4 or ( j ) SCF FBXL6 were used as a control. Proteins that were significantly enriched with CRL4 DCAF12 compared to controls are in the upper right quadrants. k, Affinity purification (AP) of a panel of cullin-RING ubiquitin ligases (CRLs). HEK293T cells were transfected with an empty vector (EV) or the indicated StrepII-FLAG-tagged CRL constructs and treated with MLN4924 for 6 hours before harvesting. Forty-eight hours after transfection, cells were harvested and lysed, and whole- cell lysates (WCL) were subjected to AP with Strep-TactinXT resin. Eluates were immunoblotted with indicated antibodies. l, Affinity purification of N-terminal STREP-FLAG-tagged DCAF12 and immunopurification of HA-tagged MCMBP, MOV10, and C-terminal degron-lacking mutants. HEK293T cells were co-transfected with StrepII-FLAG-tagged DCAF12 and either an empty vector (EV) or constructs expressing HA-tagged MOV10, MOV10ΔC, MCMBP, or MCMBPΔC. MLN4924 was added for the final 6 hours before harvest. Following lysis, whole-cell lysates (WCL) were subjected to affinity purification (AP) with Strep-TactinXT resin or anti-HA immunoprecipitation (IP) and immunoblotted as indicated. The left panel shows 1% input samples, the upper right panel shows Strep-Tactin AP, and the lower right panel shows anti-HA IP. P values were calculated by ordinary one-way ANOVA with Dunnett’s test ( b, d ); ****P<0.0001, **P<0.01, *P<0.1, n.s. (not significant) indicates P>0.1. (A.U. Arbitrary Units).

Article Snippet: For remaining experiments, siRNAs were procured from Ambion Silencer Select: DCAF1 (s18762), CDT2 (s226738), AMBRA1 (s31112), DCAF4 (s25085), DCAF6 (s31604), DCAF8 (s27089), DCAF10 (s35726), DCAF12#1 (s24628), DCAF12#2 (s24627), DCAF14 (s30012), DCAF16 (s29650), and MCMBP (s36586).

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

a, QIBC plots of naïve U2OS cells and DCAF12 knock-out U2OS cells (DCAF12-KO#1, DCAF12-KO#2) expressing doxycycline (DOX, 48 hours) inducible Strep-DCAF12 (where indicated) immunostained for MCMBP. Lines denote medians; n ≈ 9,200 cells per condition. b, Quantification of QIBC plots in a ; each bar indicates the median of mean intensity (data are mean ± s.d.; n = 2 technical replicates). c, Mean fluorescent intensity (MFI) of cytoplasmic MCMBP under similar conditions as in a . Central lines of box plots denote medians, the boxes indicate the 25 th and 75 th centiles, the whiskers indicate 5 and 95 percentile values; n = 200 cells per condition. d, Quantification of the MFI of cytoplasmic MCMBP in c ; each bar indicates the median of mean intensity normalized with respect to naïve U2OS as 100 percent (data are mean ± s.d.; n = 2 technical replicates). e, RT-PCR-based analysis of MCMBP mRNA levels in naïve U2OS cells and DCAF12 knock-out U2OS cells (DCAF12- KO#1, DCAF12-KO#2) expressing DOX inducible Strep-DCAF12 (where indicated); normalized with respect to naïve U2OS cells. n = 3 technical replicates. f, StrepII-FLAG-tagged DCAF12 (SF-DCAF12) was inducible expressed in DCAF12 WT and KO U2OS cells using the Sleeping Beauty Transposon System. Where indicated, the expression of DCAF12 was induced with doxycycline for 24 h. Cells were treated with cycloheximide (CHX) for the indicated time. Whole-cell lysates were immunoblotted as indicated . g, Densitometry of selected protein staining in f ; h, U2OS DCAF12 WT or KO cells were synchronized in G1/S by a hydroxyurea treatment (2 mM; 16 hours) and then released in fresh medium for the indicated hours. Cells were collected at the indicated time and processed for western blot. Whole-cell lysates were immunoblotted as indicated . i, Densitometry of MCMBP staining in h; j, Densitometry of MCM6 protein staining in h . k, QIBC plots of MCM4-Halo U2OS cells treated with MG123 inhibitor and control siRNA (siCTRL) or siRNA against DCAF12 (siDCAF12). Lines denote medians; n ≈ 5,000 cells per condition. l, Quantification of QIBC plots in k ; each bar indicates the median of mean intensity normalized with respect to siCTRL as 100 percent (data are mean ± s.d.; n = 3 technical replicates). P values were calculated by ordinary one-way ANOVA with Tukey’s test ( b, d ) or Dunnett’s test ( l ); ***P<0.001, **P<0.01, *P<0.1, n.s. (not significant) indicates P>0.1. (A.U. Arbitrary Units).

Journal: bioRxiv

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

doi: 10.1101/2024.11.26.625391

Figure Lengend Snippet: a, QIBC plots of naïve U2OS cells and DCAF12 knock-out U2OS cells (DCAF12-KO#1, DCAF12-KO#2) expressing doxycycline (DOX, 48 hours) inducible Strep-DCAF12 (where indicated) immunostained for MCMBP. Lines denote medians; n ≈ 9,200 cells per condition. b, Quantification of QIBC plots in a ; each bar indicates the median of mean intensity (data are mean ± s.d.; n = 2 technical replicates). c, Mean fluorescent intensity (MFI) of cytoplasmic MCMBP under similar conditions as in a . Central lines of box plots denote medians, the boxes indicate the 25 th and 75 th centiles, the whiskers indicate 5 and 95 percentile values; n = 200 cells per condition. d, Quantification of the MFI of cytoplasmic MCMBP in c ; each bar indicates the median of mean intensity normalized with respect to naïve U2OS as 100 percent (data are mean ± s.d.; n = 2 technical replicates). e, RT-PCR-based analysis of MCMBP mRNA levels in naïve U2OS cells and DCAF12 knock-out U2OS cells (DCAF12- KO#1, DCAF12-KO#2) expressing DOX inducible Strep-DCAF12 (where indicated); normalized with respect to naïve U2OS cells. n = 3 technical replicates. f, StrepII-FLAG-tagged DCAF12 (SF-DCAF12) was inducible expressed in DCAF12 WT and KO U2OS cells using the Sleeping Beauty Transposon System. Where indicated, the expression of DCAF12 was induced with doxycycline for 24 h. Cells were treated with cycloheximide (CHX) for the indicated time. Whole-cell lysates were immunoblotted as indicated . g, Densitometry of selected protein staining in f ; h, U2OS DCAF12 WT or KO cells were synchronized in G1/S by a hydroxyurea treatment (2 mM; 16 hours) and then released in fresh medium for the indicated hours. Cells were collected at the indicated time and processed for western blot. Whole-cell lysates were immunoblotted as indicated . i, Densitometry of MCMBP staining in h; j, Densitometry of MCM6 protein staining in h . k, QIBC plots of MCM4-Halo U2OS cells treated with MG123 inhibitor and control siRNA (siCTRL) or siRNA against DCAF12 (siDCAF12). Lines denote medians; n ≈ 5,000 cells per condition. l, Quantification of QIBC plots in k ; each bar indicates the median of mean intensity normalized with respect to siCTRL as 100 percent (data are mean ± s.d.; n = 3 technical replicates). P values were calculated by ordinary one-way ANOVA with Tukey’s test ( b, d ) or Dunnett’s test ( l ); ***P<0.001, **P<0.01, *P<0.1, n.s. (not significant) indicates P>0.1. (A.U. Arbitrary Units).

Techniques: Knock-Out, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Western Blot, Control

Journal: bioRxiv

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

doi: 10.1101/2024.11.26.625391

Techniques: Immunoprecipitation, Western Blot, Concentration Assay, Control, Derivative Assay, Mass Spectrometry, Transfection, Protein Purification, Staining, Construct

a, Left, labeling protocol for DNA fiber experiments and a panel of representative images of ongoing replication forks per conditions as indicated. Right, replication fork speed in U2OS cells treated with indicated siRNAs. Lines represent medians; n = 500 fibers per condition. b, Left, representative images of bidirectional replication forks; right, quantification of sister fork ratio in U2OS cells following indicated siRNA treatments. In box plots, central lines denote medians, the boxes indicate the 25 th and 75 th centiles, and the whiskers indicate Tukey values; n = 50 fibers per condition. c, Quantification of the level of γH2AX in the nuclei of S phase cells after indicated siRNA treatments. Each bar indicates the median of mean intensity normalized with respect to siCTRL as 100 percent (data are mean ± s.d.; n = 3 biological replicates). See QIBC in Extended Data Fig. 7f. d, Quantification of number of RAD51 foci per cell nucleus after indicated siRNA treatments. Each bar indicates the median of the mean foci count (data are mean ± s.d.; n = 3 biological replicates). See QIBC in Extended Data Fig. 7j. e, Left, QIBC plots of MCMBP-KO, MCMBP-wt (in MCMBP-KO), MCMBP- ΔC (in MCMBP-KO) stained for γH2AX. DAPI counterstains nuclear DNA; n ≈ 5,000 cells per condition. Right, MFI of γH2AX in S phase cells based on QIBC on the left. Lines denote median, n ≈ 5,000 cells per condition. f, Representative images (left), quantification (right) of micronucleation in U2OS cells after indicated siRNA treatments (n ≈ 500 cells per condition) (data are mean ± s.d.; n = 3 biological replicates. Scale bar, 20 µm. g, Clonogenic survival of U2OS cells after 10 days of siRNA treatments, as indicated. Left, representative images; right, quantification. Each bar indicates the average of observed colonies normalized with respect to control siRNA treatment as 100 percent; n = 4 biological replicates. h, Tumor-free survival in Trp53 homozygous knockout mice group (TP53-KO) and double knockout (TP53+DCAF12-KO) mice lacking both Trp53 and Dcaf12 genes. P value was calculated by a two-tailed unpaired t -test (P = 0.0283). i, A model depicting the critical role of the MCM biogenesis pathway to support optimal levels of origin licensing. The assembly of nascent MCM complexes is regulated by the MCM biogenesis pathway (i). Precise control of the MCM biogenesis pathway ensures optimal levels of inactive nascent MCMs on chromatin (ii), which facilitates normal replication fork speed and stable genomes. Conversely, inhibition of this pathway results in a shortage of inactive nascent MCMs on chromatin, leading to accelerated fork progression and genome instability (see text for details). P values were calculated by one-way ANOVA with Tukey’s test ( a, b, c ) or Šidák’s test ( d, f, g ), ****P<0.0001, **P<0.01, *P<0.1, n.s. (not significant) indicates P>0.1. (A.U. Arbitrary Units).

Journal: bioRxiv

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

doi: 10.1101/2024.11.26.625391

Figure Lengend Snippet: a, Left, labeling protocol for DNA fiber experiments and a panel of representative images of ongoing replication forks per conditions as indicated. Right, replication fork speed in U2OS cells treated with indicated siRNAs. Lines represent medians; n = 500 fibers per condition. b, Left, representative images of bidirectional replication forks; right, quantification of sister fork ratio in U2OS cells following indicated siRNA treatments. In box plots, central lines denote medians, the boxes indicate the 25 th and 75 th centiles, and the whiskers indicate Tukey values; n = 50 fibers per condition. c, Quantification of the level of γH2AX in the nuclei of S phase cells after indicated siRNA treatments. Each bar indicates the median of mean intensity normalized with respect to siCTRL as 100 percent (data are mean ± s.d.; n = 3 biological replicates). See QIBC in Extended Data Fig. 7f. d, Quantification of number of RAD51 foci per cell nucleus after indicated siRNA treatments. Each bar indicates the median of the mean foci count (data are mean ± s.d.; n = 3 biological replicates). See QIBC in Extended Data Fig. 7j. e, Left, QIBC plots of MCMBP-KO, MCMBP-wt (in MCMBP-KO), MCMBP- ΔC (in MCMBP-KO) stained for γH2AX. DAPI counterstains nuclear DNA; n ≈ 5,000 cells per condition. Right, MFI of γH2AX in S phase cells based on QIBC on the left. Lines denote median, n ≈ 5,000 cells per condition. f, Representative images (left), quantification (right) of micronucleation in U2OS cells after indicated siRNA treatments (n ≈ 500 cells per condition) (data are mean ± s.d.; n = 3 biological replicates. Scale bar, 20 µm. g, Clonogenic survival of U2OS cells after 10 days of siRNA treatments, as indicated. Left, representative images; right, quantification. Each bar indicates the average of observed colonies normalized with respect to control siRNA treatment as 100 percent; n = 4 biological replicates. h, Tumor-free survival in Trp53 homozygous knockout mice group (TP53-KO) and double knockout (TP53+DCAF12-KO) mice lacking both Trp53 and Dcaf12 genes. P value was calculated by a two-tailed unpaired t -test (P = 0.0283). i, A model depicting the critical role of the MCM biogenesis pathway to support optimal levels of origin licensing. The assembly of nascent MCM complexes is regulated by the MCM biogenesis pathway (i). Precise control of the MCM biogenesis pathway ensures optimal levels of inactive nascent MCMs on chromatin (ii), which facilitates normal replication fork speed and stable genomes. Conversely, inhibition of this pathway results in a shortage of inactive nascent MCMs on chromatin, leading to accelerated fork progression and genome instability (see text for details). P values were calculated by one-way ANOVA with Tukey’s test ( a, b, c ) or Šidák’s test ( d, f, g ), ****P<0.0001, **P<0.01, *P<0.1, n.s. (not significant) indicates P>0.1. (A.U. Arbitrary Units).

Techniques: Labeling, Staining, Control, Knock-Out, Double Knockout, Two Tailed Test, Inhibition

Journal: iScience

Article Title: Bromodomain protein BRD8 regulates cell cycle progression in colorectal cancer cells through a TIP60-independent regulation of the pre-RC complex

doi: 10.1016/j.isci.2023.106563

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-MCMBP , Atlas Antibodies , Cat# HPA038481; RRID:AB_10794620.

Techniques: Recombinant, Plasmid Preparation, Cell Cycle Assay, CCK-8 Assay, Flow Cytometry, Viability Assay, Ubiquitin Proteomics, Expressing, Cloning, Mutagenesis, Software

Journal: iScience

Article Title: Bromodomain protein BRD8 regulates cell cycle progression in colorectal cancer cells through a TIP60-independent regulation of the pre-RC complex

doi: 10.1016/j.isci.2023.106563

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-MCMBP , Atlas Antibodies , Cat# HPA038481; RRID:AB_10794620.

Techniques: Recombinant, Plasmid Preparation, Cell Cycle Assay, CCK-8 Assay, Flow Cytometry, Viability Assay, Ubiquitin Proteomics, Expressing, Cloning, Mutagenesis, Software

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