Journal: Cell

Article Title: Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2

doi: 10.1016/j.cell.2020.03.051

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: One hundred mg of mixed protein lysate was subjected for protein digestion with trypsin using Pierce Mass Spec Sample Prep Kit for Cultured Cells (84840, Thermo Scientific), followed by phosphopeptide enrichment using High-Select TiO2 Phosphopeptide Enrichment Kit (A32993, Thermo Scientific) according to the manufacturer’s instructions.

Techniques: Western Blot, Immunohistochemistry, Flow Cytometry, Recombinant, Immunoprecipitation, IP Phosphatase Assay, Modification, Polymerization Assay, Fluorescence, Transgenic Assay, CRISPR, shRNA, Clone Assay, Plasmid Preparation, Selection, Software, Mutagenesis, Protease Inhibitor, Expressing, Cell Culture, Staining, Reporter Assay, Microscopy, Protein Quantitation, Mass Spectrometry, Sample Prep

Journal: bioRxiv

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.1101/2023.10.30.564746

Figure Lengend Snippet: (A) Volcano plot displaying the protein abundance ratios of SPARK-AID parasites treated with IAA or vehicle for 24 hours and adjusted p-values. Proteins identified as up- or down-regulated in parasites overexpressing the driver of differentiation (BFD1) are shown in blue and vermilion, respectively. In total, 4474 proteins were quantified, 3847 of which registered more than one peptide. (B) Schematic of the phosphoproteomics experiment following SPARK depletion. Intracellular parasites expressing SPARK-AID were treated with 500 µM IAA for 24, 8, 3, or 0 h and were harvested simultaneously with the TIR1 parental strain as a control. Samples were multiplexed with tandem mass tags (TMT). The same experimental design was used for SPARKEL-AID proteomics. Each experiment included two biological replicates. (C) Protein abundances of PKG, PKA-R, and PKA-C1 relative to the TIR1 parental strain after the indicated period of SPARK (thick lines) or SPARKEL (dotted lines) depletion. (D) Protein abundances of up-regulated bradyzoite genes relative to the TIR1 parental strain after the indicated period of SPARK (blue lines) or SPARKEL (purple lines) depletion. Up-regulated bradyzoite proteins were defined as those significantly increased in parasites overexpressing BFD1 and two modified Z-scores above the median in the SPARK depletion proteome. Rank-ordered plots of (E) Phosphopeptide basal dysregulation score (peptide abundance in the SPARK-AID strain without IAA treatment relative to the TIR1 parental strain) or (F) IAA-dependent score (summed peptide ratios relative to the SPARK-AID t 0 peptide abundance). Dotted lines correspond to 3.5 modified Z scores. Colored points correspond to phosphosites discussed in the main text. (G) Gene ontology (GO) enrichment of phosphoproteins exhibiting SPARK-dependent regulation. Gene ratio is the proportion of proteins with the indicated GO term divided by the total number of proteins. Significance was determined with a hypergeometric test; only GO terms with p < 0.05 are shown. Redundant GO terms were removed. Categories discussed in the main text are highlighted with colored text. (H) Gaussian mixture modeling of SPARK-dependent peptides identified by more than one PSM revealed seven kinetically resolved clusters. Individual peptides or the median ratios in each cluster are depicted by light and dark gray lines, respectively. Clusters are numbered according to their discussion in the main text. (I) Protein abundances following biotinylation and streptavidin enrichment of samples derived from parasites expressing mNG- or SPARK-TurboID fusion constructs. A pseudocount of 10 4.5 was assigned to proteins identified in only one sample. Point colors correspond to significance thresholds. Dotted lines correspond to one median absolute deviation.

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Expressing, Modification, Derivative Assay, Construct

Journal: bioRxiv

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.1101/2023.10.30.564746

Figure Lengend Snippet: (A–B) Protein and phosphopeptide abundances of PKA C1 (A) and PKA R (B) following SPARK depletion. (C) Schematic of the genetic strategy used to monitor PKA C1 and PKA R abundances following SPARK (or SPARKEL) down-regulation with IAA. (D) Immunofluorescence microscopy of parasites expressing SPARK-AID, PKA C1-mNG, and PKA R-mCherry after 0 or 24 hours of IAA treatment to degrade SPARK. Parasites and nuclei were stained with GAP45 and Hoechst 33342, respectively. GAP45 staining and mNG or mCherry staining were normalized to vehicle-treated tagged samples. (E, F) Flow cytometry analysis of parasites expressing PKA C1-mNG, PKA R-mCherry, and SPARK-AID or SPARKEL-AID, respectively, after the indicated period of IAA treatment. The dotted line centers the mode of the vehicle-treated sample. Traces were normalized by unit area. (G) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKA C1 targets, as defined in the text and methods, are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. (H) Heat map of the abundance ratios of candidate PKA C1 targets following SPARK depletion. PKA R depletion results in up-regulation of PKA C1, and candidate PKA C1 targets therefore have positive abundance ratios following PKA R down-regulation.

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Immunofluorescence, Microscopy, Expressing, Staining, Flow Cytometry, Derivative Assay

Journal: bioRxiv

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.1101/2023.10.30.564746

Figure Lengend Snippet: Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PP1 targets, as defined in the text and methods, are shown in blue. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. PP1 proteome data was obtained from .

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.1101/2023.10.30.564746

Figure Lengend Snippet: (A) Protein and phosphopeptide abundances of PKG following SPARK depletion. (B) A23187-stimulated egress assays performed at different concentrations of compound 1 after TIR1 and SPARK-AID parasites had been treated with IAA for 24 hours. Curves were fit to the average values of six replicates and were compared with an extra sum of squares F test. (C) The IC50 values of each strain for compound 1; each point represents a biological replicate. Significance was assessed with a two-tailed t -test. (D) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKG targets are shown in green. The distributions and p -values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. (E) Heat map of the abundance ratios of candidate PKG targets following SPARK depletion.

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.1101/2023.10.30.564746

Figure Lengend Snippet: (A) Protein abundances from immunopurified PKA C3-mNG-AID lysates or an untagged control strain. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 10 4.5 . Point colors correspond to significance thresholds. (B) Immunoblot of parasites expressing SPARK-V5-AID-HA and PKA C3-mNG after 24 hours of IAA treatment. ALD1 serves as a loading control. Band intensity normalized to the dual-tagged strain is shown in (C) from 3 replicates. (D) Flow cytometry analysis of SPARK-AID parasites expressing PKA C3-mNG treated with IAA for the indicated number of hours. Traces are representative of two biological replicates. The dotted line centers the mode of the vehicle-treated sample. Traces were normalized by unit area. (E–F) Fixed, intracellular SPARK-AID/PKA C3-mNG parasites visualized by immunofluorescence microscopy using the mNG epitope after the indicated period of IAA treatment (E) . The mNG signal was internally normalized to the SPARK-AID parental strain. Quantification of the PKA C3 signal intensity of three replicates is shown in (F) . (G) Phosphopeptide IAA-dependent score (summed peptide ratios relative to the PKA C3-AID t 0 peptide abundance). Dotted lines correspond to 3.5 modified Z scores. Colored points correspond to phosphosites discussed in the main text. (H) Gaussian mixture modeling of PKA C3-dependent peptides identified by more than one PSM revealed three kinetically resolved clusters. Individual peptides or the median ratios in each cluster are depicted by light and dark gray lines, respectively. Clusters are numbered according to their discussion in the main text. (I) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKA C3 targets are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. (J) Heat map of the abundance ratios of candidate PKA C3 targets following SPARK depletion. (K) Heat map of the abundance ratios of dense granule proteins following SPARK depletion, as discussed in the text.

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Modification, Western Blot, Expressing, Flow Cytometry, Immunofluorescence, Microscopy, Derivative Assay

Journal: Cell

Article Title: Allosteric Activators of Protein Phosphatase 2A Display Broad Antitumor Activity Mediated by Dephosphorylation of MYBL2

doi: 10.1016/j.cell.2020.03.051

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: FlowJo, LLC RRID:SCR_008520 Other Q5 Site-Directed Mutagenesis Kit New England BioLabs Cat#E0554S cOmplete Mini EDTA-free Protease Inhibitor Cocktail Sigma-Aldrich Cat#11836170001 Strep-Tactin XT Superflow IBA GMBH Cat#2-4012-001 Ni Sepharose 6 Fast Flow affinity resin GE Healthcare Cat# GE17-5318-01 RNeasy Mini Kit QIAGEN Cat#74104 PrestoBlue Cell Viability Reagent Thermo Fisher Scientific Cat# A13261 SpectraMax M5 Microplate reader Molecular Devices LLC Cat#M5 SuperSignal West Femto Maximum Sensitivity substrate Thermo Fisher Scientific Cat#34095 ImageQuant LAS4000 GE Healthcare Life Sciences Cat#28955810 RPMI 1640 Medium Thermo Fisher Scientific Cat#11875093 Alpha-minimum essential media 1x Glutamax-1 GIBCO Cat#32571-036 Human AB serum Sigma Cat#H4522 Insulin-Transferrin-Selenium GIBCO Cat#41100-045 CD34 MicroBead Kit, human Miltenyi Biotec Cat#130-046-702 StemMACS HSC Expansion Media XF Miltenyi Biotec Cat#130-100-473 StemMACS HSC Expansion Cocktail, human Miltenyi Biotec Cat#130-100-843 Amaxa 4D-Nucleofector Lonza Cat#AAF-1002B Multiple Lentiviral Expression System Kit Albers et al., 2015 Addgene Kit # 1000000060 FACS cell sorter BD Biosciences Cat#BD FACSAria III ReverTra Ace® qPCR RT Master Mix TOYOBO Cat#FSQ-201 ESF 921 Insect Cell Culture medium Expression Systems Cat#96-001-01 SF-900 II SFM medium Thermo Fisher Scientific Cat#10902088 ANTI-FLAG M2 affinity gel Sigma-Aldrich Cat#A2220 SuperBlue Ultra Coomassie stain Protea Biosciences Cat#SB-G250X-KIT FACS analyzer BD Biosciences Cat#BD FACS LSR II FACS analyzer BD Biosciences Cat# BD LSRFortessa Dual-Luciferase® Reporter Assay System Promega Cat#E1910 FITC Annexin V Apoptosis Detection Kit with PI BioLegend Cat#640914 BSA Sigma-Aldrich Cat#A3311 DMEM - Dulbecco’s Modified Eagle Medium Thermo Fisher Scientific Cat#11960069 Acetocarmine solution TCI Chemicals Cat#A0050 Fluorescence Microscope Carl Zeiss Microscopy GmbH Cat#Zeiss Axio Observer Inverted Fluorescence Microscope PBS Thermo Fisher Scientific Cat#10010023 SILAC Protein Quantitation Kit (LysC), RPMI 1640 Thermo Fisher Scientific Cat#A33971 Pierce Mass Spec Sample Prep Kit for Cultured Cells Thermo Fisher Scientific Cat#84840 High-Select TiO2 Phosphopeptide Enrichment Kit Thermo Fisher Scientific CatA32993 FBS Thermo Fisher Scientific Cat#16000044 Open in a separate window KEY RESOURCES TABLE list-behavior=unordered prefix-word= mark-type=disc max-label-size=0 Phenothiazine analog iHAP1 activates PP2A-B56ε and potently kills malignant cells iHAP1 does not inhibit dopamine signaling or cause prohibitive neurologic toxicity PP2A-B56ε dephosphorylates MYBL2-Ser241, causing prometaphase arrest with apoptosis Other PP2A activators, SMAPs, activate PP2A-B55α and target different substrates

Techniques: Western Blot, Immunohistochemistry, Flow Cytometry, Recombinant, Immunoprecipitation, IP Phosphatase Assay, Modification, Polymerization Assay, Fluorescence, Transgenic Assay, CRISPR, shRNA, Clone Assay, Plasmid Preparation, Selection, Software, Mutagenesis, Protease Inhibitor, Expressing, Cell Culture, Staining, Reporter Assay, Microscopy, Protein Quantitation, Mass Spectrometry, Sample Prep

Journal: Environmental Health Perspectives

Article Title: Integrated Studies on Male Reproductive Toxicity of Decabromodiphenyl Ethane in Zebrafish Spermatozoa Ex Vivo , Male Zebrafish in Vivo , and GC-1 Cells in Vitro

doi: 10.1289/EHP14426

Figure Lengend Snippet: Whole-proteome and phosphoproteome analysis of male zebrafish testes after in vivo exposure to 100 nM DBDPE. (A) Schematic diagram of proteome and phosphoproteome analysis. The illustration was in part created in BioRender (2024) https://BioRender.com/x32w463 . (B) Volcano plot of quantified proteins in 100 nM DBDPE-treated zebrafish testes. (C) Volcano plot of quantified phosphosites in 100 nM DBDPE-treated zebrafish testes. Upward triangle (filled in red; on the upper right corner) and upside-down triangle (filled in green; on the upper left corner) in (B) and (C) indicate up-regulation and down-regulation, respectively; gray dots indicate no significant difference. (D) Significantly enriched KEGG pathways of differentially expressed proteins (DEPs) from whole-proteome data. (E) Significantly enriched KEGG pathways of proteins harboring differentially phosphorylated sites from phosphoproteome data. The size of the dots in (D) and (E) represents the number of DEPs in the pathway, and the color of the dots represents the enrichment factors of KEGG pathway. (F) Protein–protein interaction (PPI) network after exposure to 100 nM DBDPE. The illustration was created in BioRender (2023) https://BioRender.com/o93k219 . Each large oval represents protein expression level, and smaller ovals with a tail on top represent phosphorylation levels of specific phosphorylation sites of the protein. Solid line boxes and dotted line boxes respectively indicate up-regulation and down-regulation in the proteome; gray-filled ovals indicate proteins without significant differences. Solid line circles and dotted line circles respectively indicate up-regulation and down-regulation in the phosphoproteome. Color intensity is proportional to log 2 (fold change). Data are reported in Excel Table S4. Note: DBDPE, decabromodiphenyl ethane; KEGG, Kyoto Encyclopedia of Genes and Genomes; PI3K-AKT, phosphoinositide 3-kinase/protein kinase B; SNARE, soluble N -ethylmaleimide-sensitive factor attachment protein receptor; TiO 2 , titanium dioxide.

Article Snippet: Phosphopeptides were enriched from digested protein samples using a High-Select Titanium Dioxide Phosphopeptide Enrichment kit (Thermo Fisher Scientific).

Techniques: In Vivo, Expressing, Phospho-proteomics, Titanium Dioxide

Journal: Cell Reports Medicine

Article Title: Proteogenomic analysis of lung adenocarcinoma reveals tumor heterogeneity, survival determinants, and therapeutically relevant pathways

doi: 10.1016/j.xcrm.2022.100819

Figure Lengend Snippet:

Article Snippet: Briefly, concatenated peptide fractions were vacuum dried, re-suspended in TiO 2 binding/equilibration buffer and bound to TiO 2 affinity spin columns (High-Select TiO 2 Phosphopeptide Enrichment Kit, Thermo Fisher Scientific, Inc), and sample flow-through and washes were reserved for subsequent enrichment by Fe-NTA (nitrilotriacetic acid) affinity chromatography (High-Select Fe-NTA Phosphopeptide Enrichment Kit, ThermoFisher Scientific, Inc).

Techniques: Recombinant, Blocking Assay, Stripping Membranes, Staining, Bicinchoninic Acid Protein Assay, High Throughput Screening Assay, RNA Sequencing Assay, Functional Assay, Software, Sequencing, Expressing