proq diamond phosphoprotein gel stain  (Thermo Fisher)

Journal: Cell reports

Article Title: An Aurora kinase A-BOD1L1-PP2A B56 axis promotes chromosome segregation fidelity

doi: 10.1016/j.celrep.2025.115317

Figure Lengend Snippet: (A) Experimental schematic of the proteomics screen. SW620 cells were treated for 3 days with UMK57 or the DMSO control and then synchronized by thymidine-nocodazole treatment. The DMSO control sample was treated with UMK57 for 1 h. Cells were collected and analyzed by mass spectrometry. (B) SW620 cells were prepared as in (A) but re-plated on glass coverslips instead of synchronization. After 24 h, cells were fixed and stained for Hec1 and DAPI, and the frequency of lagging chromosomes was measured. Statistical significance was calculated using Dunnett’s multiple-comparison test. Representative images from 3 independent experiments are shown. The images were adjusted evenly for brightness and contrast. Scale bar, 10 μm. * p < 0.05. (C) SW620 cells were prepared as in (A) and analyzed by mass spectrometry. The number of peptides and proteins detected in the screens are shown. (D) Proteins from the phospho-proteomics screen that were significantly changed ( p < 0.1) by Student’s t test were analyzed for gene ontology. The data were then transferred to Prism software and plotted. (E) Proteins from the phospho-proteomics screen that were significantly changed ( p < 0.1) by Student’s t test were analyzed by PhosphositePlus for sequence motif enrichment and plotted. (F) Volcano plot analysis of the phospho-proteomics screen results. Proteins of potential interest are highlighted in orange. Statistical significance was calculated using two-tailed t tests. (G) Analysis of overlap between the phospho-proteomics study presented here and a previous study. Proteins with changes in phosphorylation greater than 4-fold caused by AurKA or AurKB inhibitors were considered targets of AurKA or AurKB. The list of Aurora A and Aurora B targets was compared to the list of proteins from the phospho-proteomics screen that had significantly changed phosphorylation sites ( p < 0.1) by Student’s t test. (H) Schematic of the FAM44 protein family, showing the Bod1-similar domain and potential AurKA sites on BOD1L1. Scales are approximate. (I) Amino acid sequence alignment of BOD1L1 orthologs from residues 2,933–2,992 (relative to the human sequence). Residues highlighted in pink indicate Aurora kinase-defining motifs. Residues highlighted in yellow indicate potential phosphorylation sites. (J) SW620 cells were synchronized and treated with DMSO or MLN8237. BOD1L1 or control immunoprecipitations were performed, and the entirety was separated by SDS-PAGE and stained with ProQ Diamond phosphoprotein stain, imaged, and subsequently stained with Coomassie blue and re-imaged. Two independent experiments were performed with similar results.

Article Snippet: ProQ Diamond phosphoprotein stain , ThermoFisher Scientific , Cat# P33301.

Techniques: Control, Mass Spectrometry, Staining, Comparison, Phospho-proteomics, Software, Sequencing, Two Tailed Test, SDS Page

Journal: Cell reports

Article Title: An Aurora kinase A-BOD1L1-PP2A B56 axis promotes chromosome segregation fidelity

doi: 10.1016/j.celrep.2025.115317

Figure Lengend Snippet: (A) Experimental schematic to identify BOD1L1-interacting proteins. SW620 cells were synchronized, and BOD1L1 was then immunoprecipitated and purified along with interacting proteins. The samples were then separated by SDS-PAGE. The entire lanes were excised, cut into 5 pieces, and analyzed by mass spectrometry. The experiment was performed once. (B) Selected interacting proteins detected in the control immunoprecipitation and BOD1L1 immunoprecipitations showing the number of detected peptides. (C) SW620 cells were prepared as in (A), except that they were analyzed by western blot. BOD1L1 was immunoprecipitated, and the purified proteins were then separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted as indicated. The panels were adjusted for brightness and contrast. An experiment representative of two independent biological repeats is shown. (D) SW620 mitotic nuclear cell extracts were incubated with GST or GST-BOD1L fragments. Complexes were then isolated by glutathione Sepharose and analyzed by western blot. The blot shown is representative of 2 independent experiments. (E) Alphafold Multimer model of BOD1L1 F6 bound to PPP2R5D, showing the acidic patch on PPP2R5D in red and the basic stretches of BOD1L1 in blue. (F) HEK293T cells were transfected with 3×FLAG-PPP2R5C or empty vector. 48 h following transfection, the cells were harvested and lysed. 3×FLAG-PPP2R5C was then purified by immunoprecipitation. GST control or BOD1L1 F6 was then added, along with GFP-KIF4A 799–1,232 phosphorylated in vitro by GFP-His-Aurora A. The samples were then incubated at 37°C until the reaction was stopped by addition of 4× sample buffer. The reaction was then run on SDS-PAGE gel, which was then stained with ProQ-Diamond, imaged, stained with Coomassie, and re-imaged. Band intensities were measured using Fiji using an adjacent empty lane for background intensity, which was subtracted. The band intensities were then normalized and plotted. Three independent biological experiments were performed, and the data from each time point were averaged. A one-phase exponential decay curve was then fitted to the data.

Article Snippet: ProQ Diamond phosphoprotein stain , ThermoFisher Scientific , Cat# P33301.

Techniques: Immunoprecipitation, Purification, SDS Page, Mass Spectrometry, Control, Western Blot, Membrane, Incubation, Isolation, Transfection, Plasmid Preparation, In Vitro, Staining

Journal: Cell reports

Article Title: An Aurora kinase A-BOD1L1-PP2A B56 axis promotes chromosome segregation fidelity

doi: 10.1016/j.celrep.2025.115317

Figure Lengend Snippet:

Article Snippet: ProQ Diamond phosphoprotein stain , ThermoFisher Scientific , Cat# P33301.

Techniques: Virus, Recombinant, Staining, shRNA, Cloning, Plasmid Preparation, Software

Journal: The Journal of Biological Chemistry

Article Title: Influence of native thin filament type on the regulation of atrial and ventricular myosin motor activity

doi: 10.1016/j.jbc.2024.107854

Figure Lengend Snippet: Phosphorylation levels of proteins in myosin and thin filament samples obtained from atrium and ventricle. A , ProQ Diamond stained SDS-PAGE gel depicting protein phosphorylation. The left image shows the original and the right one a contrast-enhanced image of the gel. Phosphorylated forms of TnT, Tm, TnI, and MLC2a (shown with an asterisk ) are discernible. B , corresponding Coomassie stained gel shows the total protein content. C , densitometric analysis using the ProQ/Coomassie ratio reveals no significant differences in the phosphorylation levels of aTF and vTF proteins TnT, Tm, and TnI in two different preparations. The statistical significance was checked using the two-tailed unpaired t test.

Article Snippet: The gel was stained with ProQ Diamond (Molecular Probes, P33301) and imaged with 45 s exposure time (GE Healthcare, ImageQuant LAS 4000).

Techniques: Staining, SDS Page, Two Tailed Test