anti bubr1 rabbit antibody  (Bethyl)

Journal: bioRxiv

Article Title: Mitotic CDK4/6 activity sustains spindle checkpoint signalling to prevent mitotic slippage and genomic instability

doi: 10.1101/2025.09.13.675969

Figure Lengend Snippet: a, HCT116 cells were synchronised in prometaphase using thymidine-nocodazole treatment, followed by 9 hours of nocodazole incubation. Cells were treated with Palbo or a CDK1-specific inhibitor, RO-3306 (CDK1i, 10 µM), with or without MG132. Samples were collected at 3 and 7 hours after treatment for flow cytometry and immunoblot analysis. Mitotic exit (decrease in pH3-positive cells) induced by Palbo, but not by CDK1i, was effectively blocked by proteasome inhibition. Data are means ± s.d. (N = 3); two-way ANOVA. Western blot analysis shows retention of highly phosphorylated Cdc27 and CycB1 in MG132-treated cells. b, Cells were synchronised in prometaphase as above and treated with Palbo or vehicle control in the presence or absence of the APC/C inhibitor, proTAME (25 µM). Flow cytometry analysis revealed that proTAME blocked mitotic exit induced by CDK4/6 inhibition, as evidenced by retention of pH3-positive cells with high CycB1 levels. c, HCT116 cells were arrested in mitosis using nocodazole and co-treated with MG132 to prevent mitotic exit. BubR1 and centromere marker CENP-C were visualised by immunofluorescence. BubR1 kinetochore localisation decreased over time upon Palbo treatment, while it remained stable in control conditions. Alisertib (1 µM) served as a positive control for SAC inactivation. Scale bar: 10 µm. Pearson correlation coefficients (PCC) quantify BubR1/CENP-C co-localisation; one-way ANOVA. n = 20, 100, 100, 100 (-); 20, 99, 100, 100 (+Palbo); 21, 100, 98, 100 (+Alisertib), at T0, T1, T2, T3 in a single experient. d, CDK4/6 inhibition reduces KNL1 MELT phosphorylation (pMELT) and BubR1 phosphorylation. Left: Immunoblots show decreased pMELT and BubR1 phosphorylation in cells treated with Palbo, Abema (5 µM), or alisertib in the presence of MG132, correlating with SAC inactivation. Right: Immunofluorescence shows partial reduction of kinetochore pMELT signals after Palbo or Abema treatment. Scale bar: 10 µm.

Article Snippet: Antibodies used for immunofluorescence included Lamin A/C (Abcam ab238303), β-Tubulin (ABclonal A12289), BubR1 (Abclonal A1775), CENP-C (MBL PD030), phospho-Histone H3 Ser10 (ABclonal AP0002), phospho-KNL1 MELT (CST #40758), CDK4 (Santa Cruz sc-23896), and CDK6 (Santa Cruz sc-7961), and Cyclin D1 (Santa Cruz sc-8396).

Techniques: Incubation, Flow Cytometry, Western Blot, Inhibition, Control, Marker, Immunofluorescence, Positive Control, Phospho-proteomics

Journal: bioRxiv

Article Title: Mitotic CDK4/6 activity sustains spindle checkpoint signalling to prevent mitotic slippage and genomic instability

doi: 10.1101/2025.09.13.675969

Figure Lengend Snippet: a, Schematic of the experimental workflow for phosphoproteomic analysis. HCT116 cells were synchronised at prometaphase by 9-hour nocodazole and MG132 treatment after thymidine release (T0). Cells were exposed to 1 µM or 5 µM Palbo or control (-) for 2 hours (T2) and harvested for phosphoproteomic analysis. BubR1 dissociation from kinetochores and cell cycle profiles were confirmed as indicators of CDK4/6 inhibition efficiency. b, Summary of identified phosphorylation sites, peptides, and proteins across all samples. Sites and proteins that were comparable between treatments are shown. c, Numbers of differentially phosphorylated sites (upregulated and downregulated) and proteins in 1 µM and 5 µM Palbo-treated cells compared to controls (fold change > 1.5 or < 0.667, p < 0.05). d, Heatmap of global phosphorylation profiles across all samples, showing changes induced by 1 µM and 5 µM Palbo treatment. e, Established CDK4/6 substrates, RB1 and RBL1, showed significant reduction in phosphorylation upon treatment with Palbo treatment. f, Motif enrichment analysis of differentially phosphorylated sites revealed significant enrichment of the pS/T-P motif, characteristic of CDK4/6 substrates. g, Venn diagram illustrating chromosome segregation-related proteins among significantly downregulated phosphoproteins upon 1 µM or/and 5 µM Palbo treatment (fold change < 0.5, p < 0.05). h, Volcano plots showing differentially phosphorylated proteins associated with chromosome segregation in 1 µM and 5 µM Palbo treatments compared to controls. Key SAC-related proteins are highlighted. i, Putative CDK4/6 phosphorylation sites identified on SAC-related proteins, CENP-E, CENP-F, BUB1, ZW10, INCENP, and NUMA1, with significant reduction in phosphorylation upon Palbo treatment.

Article Snippet: Antibodies used for immunofluorescence included Lamin A/C (Abcam ab238303), β-Tubulin (ABclonal A12289), BubR1 (Abclonal A1775), CENP-C (MBL PD030), phospho-Histone H3 Ser10 (ABclonal AP0002), phospho-KNL1 MELT (CST #40758), CDK4 (Santa Cruz sc-23896), and CDK6 (Santa Cruz sc-7961), and Cyclin D1 (Santa Cruz sc-8396).

Techniques: Control, Inhibition, Phospho-proteomics

Journal: bioRxiv

Article Title: Mitotic CDK4/6 activity sustains spindle checkpoint signalling to prevent mitotic slippage and genomic instability

doi: 10.1101/2025.09.13.675969

Figure Lengend Snippet: a, In vitro kinase assays show that recombinant Cdk4–CycD1 complexes phosphorylate fragments of Bub1 (300–479), CENP-E (2452–2701), and CENP-F (2831–3114), but not Bub1 (1–150) or ZW10 (1–131). RBL1 (391–972) and RB (380–928) served as positive controls. Phosphorylation was detected using ATP-γS with thiophosphate ester immunoblotting. Substrate input was validated using anti-His tag antibodies. b-d, Cdk4 phosphorylation of individual SAC components: b, CENP-E 2452–2701 showed robust phosphorylation. c, CENP-F 2831–3114 was similarly phosphorylated. d, ZW10 1–131 was not phosphorylated. e, Mass spectrometry analysis identifies serine 2654 (S2654) as the CDK4 phosphorylation site within the CENP-E 2452–2701 fragment. The annotated spectrum shows y- and b-ions confirming phosphorylation at S2654. f, In vitro phosphorylation assays with wild-type (WT) and S2654A mutant CENP-E fragments show abolished phosphorylation in the S2654A mutant, confirming S2654 as the CDK4-specific site. g, Phosphoproteomic analysis of Palbo-treated mitotic cells identifies S2654 among phosphorylation sites significantly reduced upon CDK4 inhibition (*: p < 0.05). h, Schematic representation of the CENP-E domain structure, showing motor, rod, and tail regions. The tail, enriched with S/T-P motifs, contains S2654, a CDK4 phosphorylation site crucial for BubR1 recruitment to kinetochores and SAC maintenance.

Article Snippet: Antibodies used for immunofluorescence included Lamin A/C (Abcam ab238303), β-Tubulin (ABclonal A12289), BubR1 (Abclonal A1775), CENP-C (MBL PD030), phospho-Histone H3 Ser10 (ABclonal AP0002), phospho-KNL1 MELT (CST #40758), CDK4 (Santa Cruz sc-23896), and CDK6 (Santa Cruz sc-7961), and Cyclin D1 (Santa Cruz sc-8396).

Techniques: In Vitro, Recombinant, Phospho-proteomics, Western Blot, Mass Spectrometry, Mutagenesis, Inhibition