Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Linear illustration of human BAG3 protein sequence with reported p-sites annotated. (B) Co-IP of FLAG-BAG3 from HEK293 cells revealed significantly enriched proteins, assessed using the rankprod method (n = 4), with a minimum fivefold increase and a P -value ≤ 0.05. Enriched proteins in the sample are marked in respective colors for subcategories. Selected hits are annotated with their gene name. A false discovery rate of 5% is indicated by a dashed line. (C) Enriched phosphatases and respective phosphatase regulators from the BAG3 co-IP sample were assigned to their respective (super-)families of human phosphatases. The phosphoprotein phosphatase family is colored; other Ser/Thr-specific phosphatases are in shades of gray. Gene names are listed next to the corresponding assigned phosphatase identifications. PPMs, metal-dependent protein phosphatases; PSTPs, protein serine/threonine-specific phosphatases. (A, D) Gene ontology enrichment analysis of significantly enriched genes from the BAG3 co-IP sample (n = 277) in (A). The 10 most abundant biological process GO terms are displayed as bars, ranked based on protein counts. Biological processes related to BAG3’s role in protein homeostasis are presented in blue. (E) Gene ontology enrichment analysis of the co-IP sample compared with overall human gene expression, displayed as a heatmap. Fold enrichment was calculated for the BAG3 enriched, not enriched, and total co-IP sets, with overall GO categories annotated at the respective cluster. Statistical significance was determined using Fisher’s test ( P -value < 0.05) with the Bonferroni correction implemented in the PANTHER database. Non-significant changes are colored gray within the heatmap.

Article Snippet: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

Techniques: Sequencing, Co-Immunoprecipitation Assay, Gene Expression

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) A7r5 smooth muscle cells were treated with increasing concentrations of phosphoprotein phosphatase inhibitors CalA or OA as indicated, or DMSO as control. After 20 min of incubation, cells were lysed, and modulator effects on BAG3-pS136 were determined with immunoblots. The quantification is presented as a bar plot, with the mean depicted with error bars that represent the SEM based on five independent experiments. Statistical significance between inhibitors was determined with t test and P -values are displayed as stars (* P = 0.020, *** P < 0.001). (B, C) Michaelis–Menten kinetic parameters of PP1c were determined against the indicated mono- or bisphosphorylated peptides. Error bars represent the SEM of three independent replicates with technical duplicates. k cat / K m was calculated by comparison with a phosphate standard curve. (D) Overexpressed FLAG-BAG3 from transiently transfected HEK293 cells was single-step affinity immobilized with anti-FLAG beads and treated with PP1c for 30 min. Immunoblot quantification depicts the mean of four independent experiments, the error bar represents the SD, with P -values obtained from a t tests (two-tailed, paired, **** P < 0.001). (E) Monitoring of BAG3-pS136 dephosphorylation in A7r5 lysate upon incubation with PP1c, PP5, or without phosphatase as a control treatment for the indicated incubation time. Endogenous phosphatases were inhibited by the addition of 20 nM CalA. Quantification depicts the results of four independent experiments with P -values obtained from two-way ANOVA with Sidak correction (** P = 0.006 [PP1c], ** P = 0.005 [PP5], **** P < 0.001). Mean is shown and error bars represent the SEM. Source data are available for this figure.

Article Snippet: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

Techniques: Control, Incubation, Western Blot, Comparison, Transfection, Two Tailed Test, De-Phosphorylation Assay

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Illustrative overview of the small molecule and peptide modulator screen to tune PP1 activity in cells. HEK293 cells transiently expressing FLAG-BAG3 were treated as illustrated, lysed, and captured by a single-step affinity enrichment using anti-FLAG beads. Samples were analyzed by immunoblots. Quantification depicts results of three independent experiments with P -values obtained from t tests (two-tailed). Mean is shown with replicates as scatter plot and error bars represent the SD (* P = 0.015, *** P < 0.001). (B) A7r5 smooth muscle cells were incubated with increasing concentrations of Tautomycetin as indicated. Immunoblots of lysate were used to determine the sensitivity of the titrated inhibitor towards BAG3-pS136. Mean is shown as a barplot with replicates as scatter plots, and error bars represent the SD of four independent experiments. Statistical significance between concentrations is determined with t test with Welch’s correction (* P < 0.05). (C) Analysis of phosphorylation-dependent binding of 14-3-3γ to FLAG-BAG3 in A7r5 muscle cells after 20-min modulation of endogenous PP1 before lysis and FLAG-IP enrichment. Results are presented as a barplot, P -values obtained from a t test (*** P = 0.0002, **** P < 0.0001). (D) Immunoblots depicting siRNA-mediated knockdown of all PP1 isoforms combined (PP1α/β/γ) or individual PP1 isoforms (PP1α, PP1β, PP1γ) separately. The displayed blots represent the analyzed protein levels of 24 or 48 h after transfection. (E) Quantification of immunoblotted lysates show level changes of PP1α/β/γ and BAG3 (n = 3 or 4). Quantification depicts results of three or four independent experiments with P -values obtained from t tests (two-tailed, paired). Error bars represent the SD. Source data are available for this figure.

Article Snippet: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

Techniques: Activity Assay, Expressing, Western Blot, Two Tailed Test, Incubation, Phospho-proteomics, Binding Assay, Lysis, Knockdown, Transfection

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Schematic illustration of the protein level assessment after treatment of HEK293 cells with pre-designed siRNA. HEK293 cells were transfected with siRNA of PP1 isoforms (PPP1CA/B/C) for 24 and 48 h. (B) Quantification of immunoblotted lysates show phosphorylation level changes of BAG3-pS136 relative to the overall BAG3 levels (n = 3 or 4). Quantification depicts results of three or four independent experiments with P -values obtained from t tests (two-tailed, paired). Error bars represent the SD. Source data are available for this figure.

Article Snippet: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

Techniques: Transfection, Phospho-proteomics, Two Tailed Test

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: Dephosphorylation of pS136 on BAG3 by PP1 leads to loss of 14-3-3 protein binding. Dephosphorylation of the p-site cluster pS284-pS291 by PP5 enables HspB8 binding, and PP5 depletion increases BAG3 levels in a CASA-dependent manner.

Article Snippet: Isoform-specific PP1 antibodies α-PP1α/β/γ (1:1,000, sc-271762/sc-365678/sc-515943) were from Santa Cruz.

Techniques: De-Phosphorylation Assay, Protein Binding, Binding Assay

Journal: bioRxiv

Article Title: Phostensin Enables Lymphocyte Integrin Activation and Population of Peripheral Lymphoid Organs

doi: 10.1101/2021.09.24.461584

Figure Lengend Snippet: (A) Silencing of PTSN expression promotes Rap1 phosphorylation. α IIb (SBP)β 3 (D119A) expressing U2-OS cells were transduced with a lentivirus encoding a shRNA against PTSN or sh-CTRL (scrambled) and then transfected with a plasmid encoding Myc-tagged Rap1 in combination with a vector encoding mCherry-tagged PTSNor empty vector. Rap1 was immunoprecipitated using an anti-Myc antibody and phosphorylation of Rap1 was revealed by an antibody recognizing phosphorylated PKA substrates (pRap1). (B) As a control, the phosphorylation of CREB was assayed in PTSN-depleted cells using an antibody that specifically recognizes the phosphorylated form of CREB. U2-OS cells were treated with 10 μM forskolin for 30 min to induce CREB phosphorylation. (C) Phosphorylation-resistant Rap1 mutant (S180A) rescues RIAM MIT complex assembly. α IIb (SBP)β 3 (D119A) expressing U2-OS cells were transduced with a lentivirus encoding a shRNA against PTSN and transfected with a bicistronic plasmid encoding Myc-tagged Rap1 in combination with Flag-RIAM. RIAM was immunoprecipitated using an anti-Flag antibody and the associated β 3 integrin was revealed by immunoblotting. (D) The KISF motif in PTSN binds to the catalytic subunit of protein phosphatase 1 (PP1c). (E) Mutation of the KISF residues into four Ala (4A) in PTSN blocks its interaction with PP1c. HEK293 cells were transfected with plasmids encoding EGFP-tagged PP1c in combination with either β-PTSN WT or (4A) mutant. PTSN was immunoprecipitated using an anti-Flag antibody prior to immunoblotting. (F-G) HEK293 cells that express constitutively active α IIb (R995A)β 3 were transduced with a lentivirus encoding a shRNA against PTSN and transfected with a plasmid encoding EGFP-tagged shRNA resistant PTSN either WT or 4A mutant. (F) 293A cells that express constitutively active α IIb (R995A)β 3 were transduced with a lentivirus encoding a shRNA against PTSN. A scrambled shRNA was used as a negative control. Cells were then transfected with a plasmid encoding DsRed-tagged shRNA resistant β-PTSN either WT or 4A mutant. Integrin activation was measured by flow cytometry using the monoclonal antibody PAC1 that specifically recognizes the activated form of α IIb β 3 . The activation index was calculated as ( F - Fo )/( Fm - Fo ), in which F is the mean fluorescence intensity (MFI) of PAC1 binding; Fo in presence of the competitive inhibitor integrilin; and Fm upon addition of the integrin-activating anti-LIBS6 antibody. Bar graphs represent mean ± SEM (n = 3 independent experiments). One-way ANOVA with Tukey post-test; ns not significant, ** p<0.01, *** p<0.001. (G) The expression of PTSN was confirmed by immunoblotting.

Article Snippet: Plasmid encoding EGFP-tagged PP1 catalytic subunit γ was obtained from Addgene (Addgene plasmid # 44225).

Techniques: Expressing, Transduction, shRNA, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Western Blot, Negative Control, Activation Assay, Flow Cytometry, Fluorescence, Binding Assay

Journal: Structure (London, England : 1993)

Article Title: KNL1 binding to PP1 and microtubules is mutually exclusive

doi: 10.1016/j.str.2018.06.013

Figure Lengend Snippet: KEY RESOURCE TABLE (uploaded separately)

Article Snippet: pRRP1B-Thio 6 -His 6 TEV-PP1 γ 7–323 , Addgene , Id 51770.

Techniques: Expressing, Recombinant, Binding Assay, Spin Down Assay, Mutagenesis, Software

Journal: Structure (London, England : 1993)

Article Title: KNL1 binding to PP1 and microtubules is mutually exclusive

doi: 10.1016/j.str.2018.06.013

Figure Lengend Snippet: (A) KNL1 domain structure, highlighting the putative protein interaction sites (microtubule, MT, blue; PP1, magenta). The PP1 binding domain is hypothesized to contain three established PP1 binding SLiMs, namely the SILK, RVxF and ΦΦ motifs, while the putative MT binding domain is positively charged. The constructs and variants examined in this study are shown below.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial Strains E.coli BL21 (DE3) RIL expression strain Agilent Cat#230140 E.coli BL21 (DE3) Gold expression strain Agilent Cat #230132 Recombinant DNA GFP-KNL-1 wt Hardened Addgene Id 45223 pET-M30-MBP-TEV-KNL1 1–150 This study N/A pET-M30-MBP-TEV-KNL1 1–80 This study N/A pET-M30-MBP-TEV-KNL1 23–80 This study N/A pET-M30-MBP-TEV-KNL1 22–80 This study N/A pET-M30-MBP-TEV-KNL1 1–80S24A This study N/A pET-M30-MBP-TEV-KNL1 1–80S24AS25A This study N/A pET-M30-MBP-TEV-KNL1 1–80S60A This study N/A pET-M30-MBP-TEV-KNL1 1–80SILK/AAAA This study N/A pET-M30-MBP-TEV-KNL1 1–80S24DS25D This study N/A pET-M30-MBP-TEV-KNL1 1–80S56D This study N/A pET-M30-MBP-TEV-KNL1 1–80S60D This study N/A pGEX-Aurora-INCENP or AUKB M. Bollean Laboratory Available on request pRRP1B-Thio 6 -His 6 TEV-PP1 γ 7–323 Addgene Id 51770 pRRP1B-Thio 6 -His 6 TEV PP1 α 7–330 Addgene Id 51768 pRRP1B-Thio 6 -His 6 TEV- PP1 α 7–300 Addgene Id 26566 Chemicals, Peptides, and Recombinant Proteins Tubulin protein ( 99% pure, Porcine brain) Cytoskeleton, Inc. Cat #T240 General Tubulin Buffer Cytoskeleton, Inc. Cat # BST06 Tubulin Glycerol Buffer Cytoskeleton, Inc. Cat # BST05 GTP Cytoskeleton, Inc. Cat # BST06 Paclitaxel Cytoskeleton, Inc. (Cat. # TXD01) Gro 7 Chaperone Takara clonetch Cat #3340 Critical Commercial Assays Microtubule Binding protein Spin Down Assay Biochem Kit Cytoskeleton, Inc. Cat # BK029 QuikChange site directed mutagenesis Kit Agilent Cat# 200524 PDB search model Kelker (2009) PDBID: 3E7A Deposited Data BMRB (NMR assignment KNL-1 1–80 and pKNL-1 1–80 This study BMRBID: 27057, 27066 PDB This study PDBID: 6CZO Software and Algorithms Topspin 3.1 and 3.2 Bruker https://www.bruker.com UCSF SPARKY Goddard et al., 2004 https://www.cgl.ucsf.edu/home/sparky/ NMR Pipe Delaglio et al., 1995 https://www.ibbr.umd.edu/nmrpipe/install.html CARA www.cara.nmr.ch www.cara.nmr.ch PHENIX Zwart et al., 2008 https://www.phenix-online.org/ COOT Emsley et al., 2010 https://www.ccp4.ac.uk Pymol Schrodinger, LLC https://www.pymol.org NITPIC Brautigam (2015) http://biophysics.swmed.edu/MBR/software.html SEDPHAT Zhao et al. 2015 http://biophysics.swmed.edu/MBR/software.html GUSSI Brautigam (2015) http://www.biophysics.bioc.cam.ac.uk/?p=736 XDS Kabsch,W (2010) http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds Open in a separate window KEY RESOURCE TABLE (uploaded separately)

Techniques: Binding Assay, Construct

Journal: Structure (London, England : 1993)

Article Title: KNL1 binding to PP1 and microtubules is mutually exclusive

doi: 10.1016/j.str.2018.06.013

Figure Lengend Snippet: Isothermal titration calorimetry (ITC) measurements of WT KNL1 and KNL1 variants with PP1.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial Strains E.coli BL21 (DE3) RIL expression strain Agilent Cat#230140 E.coli BL21 (DE3) Gold expression strain Agilent Cat #230132 Recombinant DNA GFP-KNL-1 wt Hardened Addgene Id 45223 pET-M30-MBP-TEV-KNL1 1–150 This study N/A pET-M30-MBP-TEV-KNL1 1–80 This study N/A pET-M30-MBP-TEV-KNL1 23–80 This study N/A pET-M30-MBP-TEV-KNL1 22–80 This study N/A pET-M30-MBP-TEV-KNL1 1–80S24A This study N/A pET-M30-MBP-TEV-KNL1 1–80S24AS25A This study N/A pET-M30-MBP-TEV-KNL1 1–80S60A This study N/A pET-M30-MBP-TEV-KNL1 1–80SILK/AAAA This study N/A pET-M30-MBP-TEV-KNL1 1–80S24DS25D This study N/A pET-M30-MBP-TEV-KNL1 1–80S56D This study N/A pET-M30-MBP-TEV-KNL1 1–80S60D This study N/A pGEX-Aurora-INCENP or AUKB M. Bollean Laboratory Available on request pRRP1B-Thio 6 -His 6 TEV-PP1 γ 7–323 Addgene Id 51770 pRRP1B-Thio 6 -His 6 TEV PP1 α 7–330 Addgene Id 51768 pRRP1B-Thio 6 -His 6 TEV- PP1 α 7–300 Addgene Id 26566 Chemicals, Peptides, and Recombinant Proteins Tubulin protein ( 99% pure, Porcine brain) Cytoskeleton, Inc. Cat #T240 General Tubulin Buffer Cytoskeleton, Inc. Cat # BST06 Tubulin Glycerol Buffer Cytoskeleton, Inc. Cat # BST05 GTP Cytoskeleton, Inc. Cat # BST06 Paclitaxel Cytoskeleton, Inc. (Cat. # TXD01) Gro 7 Chaperone Takara clonetch Cat #3340 Critical Commercial Assays Microtubule Binding protein Spin Down Assay Biochem Kit Cytoskeleton, Inc. Cat # BK029 QuikChange site directed mutagenesis Kit Agilent Cat# 200524 PDB search model Kelker (2009) PDBID: 3E7A Deposited Data BMRB (NMR assignment KNL-1 1–80 and pKNL-1 1–80 This study BMRBID: 27057, 27066 PDB This study PDBID: 6CZO Software and Algorithms Topspin 3.1 and 3.2 Bruker https://www.bruker.com UCSF SPARKY Goddard et al., 2004 https://www.cgl.ucsf.edu/home/sparky/ NMR Pipe Delaglio et al., 1995 https://www.ibbr.umd.edu/nmrpipe/install.html CARA www.cara.nmr.ch www.cara.nmr.ch PHENIX Zwart et al., 2008 https://www.phenix-online.org/ COOT Emsley et al., 2010 https://www.ccp4.ac.uk Pymol Schrodinger, LLC https://www.pymol.org NITPIC Brautigam (2015) http://biophysics.swmed.edu/MBR/software.html SEDPHAT Zhao et al. 2015 http://biophysics.swmed.edu/MBR/software.html GUSSI Brautigam (2015) http://www.biophysics.bioc.cam.ac.uk/?p=736 XDS Kabsch,W (2010) http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds Open in a separate window KEY RESOURCE TABLE (uploaded separately)

Techniques: Isothermal Titration Calorimetry

Journal: Structure (London, England : 1993)

Article Title: KNL1 binding to PP1 and microtubules is mutually exclusive

doi: 10.1016/j.str.2018.06.013

Figure Lengend Snippet: (A) Overlay of the KNL1 (magenta) and Inhibitor-2 (beige) SILK motifs bound to PP1.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial Strains E.coli BL21 (DE3) RIL expression strain Agilent Cat#230140 E.coli BL21 (DE3) Gold expression strain Agilent Cat #230132 Recombinant DNA GFP-KNL-1 wt Hardened Addgene Id 45223 pET-M30-MBP-TEV-KNL1 1–150 This study N/A pET-M30-MBP-TEV-KNL1 1–80 This study N/A pET-M30-MBP-TEV-KNL1 23–80 This study N/A pET-M30-MBP-TEV-KNL1 22–80 This study N/A pET-M30-MBP-TEV-KNL1 1–80S24A This study N/A pET-M30-MBP-TEV-KNL1 1–80S24AS25A This study N/A pET-M30-MBP-TEV-KNL1 1–80S60A This study N/A pET-M30-MBP-TEV-KNL1 1–80SILK/AAAA This study N/A pET-M30-MBP-TEV-KNL1 1–80S24DS25D This study N/A pET-M30-MBP-TEV-KNL1 1–80S56D This study N/A pET-M30-MBP-TEV-KNL1 1–80S60D This study N/A pGEX-Aurora-INCENP or AUKB M. Bollean Laboratory Available on request pRRP1B-Thio 6 -His 6 TEV-PP1 γ 7–323 Addgene Id 51770 pRRP1B-Thio 6 -His 6 TEV PP1 α 7–330 Addgene Id 51768 pRRP1B-Thio 6 -His 6 TEV- PP1 α 7–300 Addgene Id 26566 Chemicals, Peptides, and Recombinant Proteins Tubulin protein ( 99% pure, Porcine brain) Cytoskeleton, Inc. Cat #T240 General Tubulin Buffer Cytoskeleton, Inc. Cat # BST06 Tubulin Glycerol Buffer Cytoskeleton, Inc. Cat # BST05 GTP Cytoskeleton, Inc. Cat # BST06 Paclitaxel Cytoskeleton, Inc. (Cat. # TXD01) Gro 7 Chaperone Takara clonetch Cat #3340 Critical Commercial Assays Microtubule Binding protein Spin Down Assay Biochem Kit Cytoskeleton, Inc. Cat # BK029 QuikChange site directed mutagenesis Kit Agilent Cat# 200524 PDB search model Kelker (2009) PDBID: 3E7A Deposited Data BMRB (NMR assignment KNL-1 1–80 and pKNL-1 1–80 This study BMRBID: 27057, 27066 PDB This study PDBID: 6CZO Software and Algorithms Topspin 3.1 and 3.2 Bruker https://www.bruker.com UCSF SPARKY Goddard et al., 2004 https://www.cgl.ucsf.edu/home/sparky/ NMR Pipe Delaglio et al., 1995 https://www.ibbr.umd.edu/nmrpipe/install.html CARA www.cara.nmr.ch www.cara.nmr.ch PHENIX Zwart et al., 2008 https://www.phenix-online.org/ COOT Emsley et al., 2010 https://www.ccp4.ac.uk Pymol Schrodinger, LLC https://www.pymol.org NITPIC Brautigam (2015) http://biophysics.swmed.edu/MBR/software.html SEDPHAT Zhao et al. 2015 http://biophysics.swmed.edu/MBR/software.html GUSSI Brautigam (2015) http://www.biophysics.bioc.cam.ac.uk/?p=736 XDS Kabsch,W (2010) http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds Open in a separate window KEY RESOURCE TABLE (uploaded separately)

Techniques:

Journal: Structure (London, England : 1993)

Article Title: KNL1 binding to PP1 and microtubules is mutually exclusive

doi: 10.1016/j.str.2018.06.013

Figure Lengend Snippet: (A) Sequence of WT-KNL11–80 with the potential phosphorylation sites indicated; the residues that bind directly to PP1 are underlined. The four residues phosphorylated directly by Aurora B kinase, as observed using NMR spectroscopy, are indicated (Ser24, Ser25, Ser56 and Ser60).

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial Strains E.coli BL21 (DE3) RIL expression strain Agilent Cat#230140 E.coli BL21 (DE3) Gold expression strain Agilent Cat #230132 Recombinant DNA GFP-KNL-1 wt Hardened Addgene Id 45223 pET-M30-MBP-TEV-KNL1 1–150 This study N/A pET-M30-MBP-TEV-KNL1 1–80 This study N/A pET-M30-MBP-TEV-KNL1 23–80 This study N/A pET-M30-MBP-TEV-KNL1 22–80 This study N/A pET-M30-MBP-TEV-KNL1 1–80S24A This study N/A pET-M30-MBP-TEV-KNL1 1–80S24AS25A This study N/A pET-M30-MBP-TEV-KNL1 1–80S60A This study N/A pET-M30-MBP-TEV-KNL1 1–80SILK/AAAA This study N/A pET-M30-MBP-TEV-KNL1 1–80S24DS25D This study N/A pET-M30-MBP-TEV-KNL1 1–80S56D This study N/A pET-M30-MBP-TEV-KNL1 1–80S60D This study N/A pGEX-Aurora-INCENP or AUKB M. Bollean Laboratory Available on request pRRP1B-Thio 6 -His 6 TEV-PP1 γ 7–323 Addgene Id 51770 pRRP1B-Thio 6 -His 6 TEV PP1 α 7–330 Addgene Id 51768 pRRP1B-Thio 6 -His 6 TEV- PP1 α 7–300 Addgene Id 26566 Chemicals, Peptides, and Recombinant Proteins Tubulin protein ( 99% pure, Porcine brain) Cytoskeleton, Inc. Cat #T240 General Tubulin Buffer Cytoskeleton, Inc. Cat # BST06 Tubulin Glycerol Buffer Cytoskeleton, Inc. Cat # BST05 GTP Cytoskeleton, Inc. Cat # BST06 Paclitaxel Cytoskeleton, Inc. (Cat. # TXD01) Gro 7 Chaperone Takara clonetch Cat #3340 Critical Commercial Assays Microtubule Binding protein Spin Down Assay Biochem Kit Cytoskeleton, Inc. Cat # BK029 QuikChange site directed mutagenesis Kit Agilent Cat# 200524 PDB search model Kelker (2009) PDBID: 3E7A Deposited Data BMRB (NMR assignment KNL-1 1–80 and pKNL-1 1–80 This study BMRBID: 27057, 27066 PDB This study PDBID: 6CZO Software and Algorithms Topspin 3.1 and 3.2 Bruker https://www.bruker.com UCSF SPARKY Goddard et al., 2004 https://www.cgl.ucsf.edu/home/sparky/ NMR Pipe Delaglio et al., 1995 https://www.ibbr.umd.edu/nmrpipe/install.html CARA www.cara.nmr.ch www.cara.nmr.ch PHENIX Zwart et al., 2008 https://www.phenix-online.org/ COOT Emsley et al., 2010 https://www.ccp4.ac.uk Pymol Schrodinger, LLC https://www.pymol.org NITPIC Brautigam (2015) http://biophysics.swmed.edu/MBR/software.html SEDPHAT Zhao et al. 2015 http://biophysics.swmed.edu/MBR/software.html GUSSI Brautigam (2015) http://www.biophysics.bioc.cam.ac.uk/?p=736 XDS Kabsch,W (2010) http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds Open in a separate window KEY RESOURCE TABLE (uploaded separately)

Techniques: Sequencing, Spectroscopy

Journal: Structure (London, England : 1993)

Article Title: KNL1 binding to PP1 and microtubules is mutually exclusive

doi: 10.1016/j.str.2018.06.013

Figure Lengend Snippet: (A) SDS-PAGE showing the cosedimentation of unbound KNL11–80 (60 μM) and the KNL1:PP1 holoenzyme (60 μM).

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial Strains E.coli BL21 (DE3) RIL expression strain Agilent Cat#230140 E.coli BL21 (DE3) Gold expression strain Agilent Cat #230132 Recombinant DNA GFP-KNL-1 wt Hardened Addgene Id 45223 pET-M30-MBP-TEV-KNL1 1–150 This study N/A pET-M30-MBP-TEV-KNL1 1–80 This study N/A pET-M30-MBP-TEV-KNL1 23–80 This study N/A pET-M30-MBP-TEV-KNL1 22–80 This study N/A pET-M30-MBP-TEV-KNL1 1–80S24A This study N/A pET-M30-MBP-TEV-KNL1 1–80S24AS25A This study N/A pET-M30-MBP-TEV-KNL1 1–80S60A This study N/A pET-M30-MBP-TEV-KNL1 1–80SILK/AAAA This study N/A pET-M30-MBP-TEV-KNL1 1–80S24DS25D This study N/A pET-M30-MBP-TEV-KNL1 1–80S56D This study N/A pET-M30-MBP-TEV-KNL1 1–80S60D This study N/A pGEX-Aurora-INCENP or AUKB M. Bollean Laboratory Available on request pRRP1B-Thio 6 -His 6 TEV-PP1 γ 7–323 Addgene Id 51770 pRRP1B-Thio 6 -His 6 TEV PP1 α 7–330 Addgene Id 51768 pRRP1B-Thio 6 -His 6 TEV- PP1 α 7–300 Addgene Id 26566 Chemicals, Peptides, and Recombinant Proteins Tubulin protein ( 99% pure, Porcine brain) Cytoskeleton, Inc. Cat #T240 General Tubulin Buffer Cytoskeleton, Inc. Cat # BST06 Tubulin Glycerol Buffer Cytoskeleton, Inc. Cat # BST05 GTP Cytoskeleton, Inc. Cat # BST06 Paclitaxel Cytoskeleton, Inc. (Cat. # TXD01) Gro 7 Chaperone Takara clonetch Cat #3340 Critical Commercial Assays Microtubule Binding protein Spin Down Assay Biochem Kit Cytoskeleton, Inc. Cat # BK029 QuikChange site directed mutagenesis Kit Agilent Cat# 200524 PDB search model Kelker (2009) PDBID: 3E7A Deposited Data BMRB (NMR assignment KNL-1 1–80 and pKNL-1 1–80 This study BMRBID: 27057, 27066 PDB This study PDBID: 6CZO Software and Algorithms Topspin 3.1 and 3.2 Bruker https://www.bruker.com UCSF SPARKY Goddard et al., 2004 https://www.cgl.ucsf.edu/home/sparky/ NMR Pipe Delaglio et al., 1995 https://www.ibbr.umd.edu/nmrpipe/install.html CARA www.cara.nmr.ch www.cara.nmr.ch PHENIX Zwart et al., 2008 https://www.phenix-online.org/ COOT Emsley et al., 2010 https://www.ccp4.ac.uk Pymol Schrodinger, LLC https://www.pymol.org NITPIC Brautigam (2015) http://biophysics.swmed.edu/MBR/software.html SEDPHAT Zhao et al. 2015 http://biophysics.swmed.edu/MBR/software.html GUSSI Brautigam (2015) http://www.biophysics.bioc.cam.ac.uk/?p=736 XDS Kabsch,W (2010) http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds Open in a separate window KEY RESOURCE TABLE (uploaded separately)

Techniques: SDS Page

Journal: Structure (London, England : 1993)

Article Title: KNL1 binding to PP1 and microtubules is mutually exclusive

doi: 10.1016/j.str.2018.06.013

Figure Lengend Snippet: (A) When the SAC is activated during prophase, KNL1 is phosphorylated by MPS1 and AURB. Threonine phosphorylation of the MELT motifs by MPS1 enables the recruitment of BUB1-BUB3 and BUBR1-BUB3 complexes via BUB3, a key step in the generation of mitotic checkpoint complexes (MCCs). At the same time, the phosphorylation of KNL1 at and near its SILK and RVSF sequences by AURB prevents the binding of both microtubules and PP1 to its N-terminus.

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial Strains E.coli BL21 (DE3) RIL expression strain Agilent Cat#230140 E.coli BL21 (DE3) Gold expression strain Agilent Cat #230132 Recombinant DNA GFP-KNL-1 wt Hardened Addgene Id 45223 pET-M30-MBP-TEV-KNL1 1–150 This study N/A pET-M30-MBP-TEV-KNL1 1–80 This study N/A pET-M30-MBP-TEV-KNL1 23–80 This study N/A pET-M30-MBP-TEV-KNL1 22–80 This study N/A pET-M30-MBP-TEV-KNL1 1–80S24A This study N/A pET-M30-MBP-TEV-KNL1 1–80S24AS25A This study N/A pET-M30-MBP-TEV-KNL1 1–80S60A This study N/A pET-M30-MBP-TEV-KNL1 1–80SILK/AAAA This study N/A pET-M30-MBP-TEV-KNL1 1–80S24DS25D This study N/A pET-M30-MBP-TEV-KNL1 1–80S56D This study N/A pET-M30-MBP-TEV-KNL1 1–80S60D This study N/A pGEX-Aurora-INCENP or AUKB M. Bollean Laboratory Available on request pRRP1B-Thio 6 -His 6 TEV-PP1 γ 7–323 Addgene Id 51770 pRRP1B-Thio 6 -His 6 TEV PP1 α 7–330 Addgene Id 51768 pRRP1B-Thio 6 -His 6 TEV- PP1 α 7–300 Addgene Id 26566 Chemicals, Peptides, and Recombinant Proteins Tubulin protein ( 99% pure, Porcine brain) Cytoskeleton, Inc. Cat #T240 General Tubulin Buffer Cytoskeleton, Inc. Cat # BST06 Tubulin Glycerol Buffer Cytoskeleton, Inc. Cat # BST05 GTP Cytoskeleton, Inc. Cat # BST06 Paclitaxel Cytoskeleton, Inc. (Cat. # TXD01) Gro 7 Chaperone Takara clonetch Cat #3340 Critical Commercial Assays Microtubule Binding protein Spin Down Assay Biochem Kit Cytoskeleton, Inc. Cat # BK029 QuikChange site directed mutagenesis Kit Agilent Cat# 200524 PDB search model Kelker (2009) PDBID: 3E7A Deposited Data BMRB (NMR assignment KNL-1 1–80 and pKNL-1 1–80 This study BMRBID: 27057, 27066 PDB This study PDBID: 6CZO Software and Algorithms Topspin 3.1 and 3.2 Bruker https://www.bruker.com UCSF SPARKY Goddard et al., 2004 https://www.cgl.ucsf.edu/home/sparky/ NMR Pipe Delaglio et al., 1995 https://www.ibbr.umd.edu/nmrpipe/install.html CARA www.cara.nmr.ch www.cara.nmr.ch PHENIX Zwart et al., 2008 https://www.phenix-online.org/ COOT Emsley et al., 2010 https://www.ccp4.ac.uk Pymol Schrodinger, LLC https://www.pymol.org NITPIC Brautigam (2015) http://biophysics.swmed.edu/MBR/software.html SEDPHAT Zhao et al. 2015 http://biophysics.swmed.edu/MBR/software.html GUSSI Brautigam (2015) http://www.biophysics.bioc.cam.ac.uk/?p=736 XDS Kabsch,W (2010) http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds Open in a separate window KEY RESOURCE TABLE (uploaded separately)

Techniques: Binding Assay

Journal: Structure (London, England : 1993)

Article Title: KNL1 binding to PP1 and microtubules is mutually exclusive

doi: 10.1016/j.str.2018.06.013

Figure Lengend Snippet: KEY RESOURCE TABLE (uploaded separately)

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial Strains E.coli BL21 (DE3) RIL expression strain Agilent Cat#230140 E.coli BL21 (DE3) Gold expression strain Agilent Cat #230132 Recombinant DNA GFP-KNL-1 wt Hardened Addgene Id 45223 pET-M30-MBP-TEV-KNL1 1–150 This study N/A pET-M30-MBP-TEV-KNL1 1–80 This study N/A pET-M30-MBP-TEV-KNL1 23–80 This study N/A pET-M30-MBP-TEV-KNL1 22–80 This study N/A pET-M30-MBP-TEV-KNL1 1–80S24A This study N/A pET-M30-MBP-TEV-KNL1 1–80S24AS25A This study N/A pET-M30-MBP-TEV-KNL1 1–80S60A This study N/A pET-M30-MBP-TEV-KNL1 1–80SILK/AAAA This study N/A pET-M30-MBP-TEV-KNL1 1–80S24DS25D This study N/A pET-M30-MBP-TEV-KNL1 1–80S56D This study N/A pET-M30-MBP-TEV-KNL1 1–80S60D This study N/A pGEX-Aurora-INCENP or AUKB M. Bollean Laboratory Available on request pRRP1B-Thio 6 -His 6 TEV-PP1 γ 7–323 Addgene Id 51770 pRRP1B-Thio 6 -His 6 TEV PP1 α 7–330 Addgene Id 51768 pRRP1B-Thio 6 -His 6 TEV- PP1 α 7–300 Addgene Id 26566 Chemicals, Peptides, and Recombinant Proteins Tubulin protein ( 99% pure, Porcine brain) Cytoskeleton, Inc. Cat #T240 General Tubulin Buffer Cytoskeleton, Inc. Cat # BST06 Tubulin Glycerol Buffer Cytoskeleton, Inc. Cat # BST05 GTP Cytoskeleton, Inc. Cat # BST06 Paclitaxel Cytoskeleton, Inc. (Cat. # TXD01) Gro 7 Chaperone Takara clonetch Cat #3340 Critical Commercial Assays Microtubule Binding protein Spin Down Assay Biochem Kit Cytoskeleton, Inc. Cat # BK029 QuikChange site directed mutagenesis Kit Agilent Cat# 200524 PDB search model Kelker (2009) PDBID: 3E7A Deposited Data BMRB (NMR assignment KNL-1 1–80 and pKNL-1 1–80 This study BMRBID: 27057, 27066 PDB This study PDBID: 6CZO Software and Algorithms Topspin 3.1 and 3.2 Bruker https://www.bruker.com UCSF SPARKY Goddard et al., 2004 https://www.cgl.ucsf.edu/home/sparky/ NMR Pipe Delaglio et al., 1995 https://www.ibbr.umd.edu/nmrpipe/install.html CARA www.cara.nmr.ch www.cara.nmr.ch PHENIX Zwart et al., 2008 https://www.phenix-online.org/ COOT Emsley et al., 2010 https://www.ccp4.ac.uk Pymol Schrodinger, LLC https://www.pymol.org NITPIC Brautigam (2015) http://biophysics.swmed.edu/MBR/software.html SEDPHAT Zhao et al. 2015 http://biophysics.swmed.edu/MBR/software.html GUSSI Brautigam (2015) http://www.biophysics.bioc.cam.ac.uk/?p=736 XDS Kabsch,W (2010) http://homes.mpimf-heidelberg.mpg.de/~kabsch/xds Open in a separate window KEY RESOURCE TABLE (uploaded separately)

Techniques: Expressing, Recombinant, Binding Assay, Spin Down Assay, Mutagenesis, Software

Journal: eLife

Article Title: Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

doi: 10.7554/eLife.25366

Figure Lengend Snippet: Mitotic progression of S2 cells depleted of PP1-87B co-expressing mCherry-α-Tubulin (red) and Mad1-EGFP (green) was monitored by spinning disk confocal microscopy. Video illustrates the prolonged metaphase duration with Mad1-EGFP present at kinetochores of bioriented chromosomes in PP1-87B depleted cells. Each frame represents a maximal intensity projection acquired every 60 s. NEB corresponds to time 0:0. Time is shown minutes. DOI: http://dx.doi.org/10.7554/eLife.25366.007

Article Snippet: Recombinant human Mps1/TTK, PP1-γ and λ-Phosphatase were purchased from SignalChem, MRC-PPU Reagents (University of Dundee) and New England Biolabs, respectively.

Techniques:

Journal: eLife

Article Title: Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

doi: 10.7554/eLife.25366

Figure Lengend Snippet: Mitotic progression of S2 cells co-depleted of PP1-87B and Mps1 co-expressing mCherry-α-Tubulin (red) and Mad1-EGFP (green) was monitored by spinning disk confocal microscopy. Video illustrates that Mps1 depletion abolishes the prolonged metaphase delay and Mad1-EGFP accumulation at bioriented kinetochores observed in PP1-87B depleted cells. Each frame represents a maximal intensity projection acquired every 60 s. NEB corresponds to time 0:0. Time is shown minutes. DOI: http://dx.doi.org/10.7554/eLife.25366.008

Article Snippet: Recombinant human Mps1/TTK, PP1-γ and λ-Phosphatase were purchased from SignalChem, MRC-PPU Reagents (University of Dundee) and New England Biolabs, respectively.

Techniques:

Journal: eLife

Article Title: Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

doi: 10.7554/eLife.25366

Figure Lengend Snippet: The Video illustrates ISI imprinting and progressive contrast fade-out of GFP–α-tubulin in a PP1-87B depleted metaphase spindle. The observed speckle contrast fadeout of tubulin is due to microtubule turnover. The time between frames is variable and is presented on the corresponding excel source data file. DOI: http://dx.doi.org/10.7554/eLife.25366.010

Article Snippet: Recombinant human Mps1/TTK, PP1-γ and λ-Phosphatase were purchased from SignalChem, MRC-PPU Reagents (University of Dundee) and New England Biolabs, respectively.

Techniques:

Journal: eLife

Article Title: Protein Phosphatase 1 inactivates Mps1 to ensure efficient Spindle Assembly Checkpoint silencing

doi: 10.7554/eLife.25366

Figure Lengend Snippet: S2 cells depleted of CENP-C expressing mCherry-α-Tubulin (red) and EGFP-Mps1 K231A/F234A (green) monitored by spinning disk confocal microscopy. Video illustrates that impairing the interaction between Mps1 and PP1-87B delays mitotic exit in kinetochore-null cells. Each frame represents a maximal intensity projection acquired every 4 min. NEB corresponds to time 0:0. Time is shown minutes. DOI: http://dx.doi.org/10.7554/eLife.25366.032

Article Snippet: Recombinant human Mps1/TTK, PP1-γ and λ-Phosphatase were purchased from SignalChem, MRC-PPU Reagents (University of Dundee) and New England Biolabs, respectively.

Techniques: