Journal: Cell

Article Title: Structural insights into the process of GPCR-G protein complex formation

doi: 10.1016/j.cell.2019.04.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Lambda protein phosphatase (2000 units, NEB), calf intestinal phosphatase (10 units, NEB), and Antarctic phosphatase (5 units, NEB) were added together with 1 mM manganese chloride, followed by a 1 h incubation at 4 °C.

Techniques: Recombinant, Expressing, Protease Inhibitor, Plasmid Preparation, Software

Journal: Microbiology Spectrum

Article Title: Duck Enteritis Virus Protein Kinase US3 Inhibits DNA Sensing Signaling by Phosphorylating Interferon Regulatory Factor 7

doi: 10.1128/spectrum.02299-22

Figure Lengend Snippet: DEV US3 phosphorylates IRF7 and blocks IRF7 dimerization and nuclear translocation. (A) IRF7-HA was cotransfected with empty vector or US3- or US3K213A-expressing plasmid into DEFs. The cells were lysed 36 h later and left untreated or treated with lambda PP prior to SDS-PAGE and Western blotting. (B) DEFs were transfected with empty vector or US3- or US3K213A-expressing plasmid. The cells were lysed 36 h later, and Western blotting was performed with anti-IRF7 antibodies to detect IRF7 phosphorylation. (C) DEFs were transfected with the indicated plasmids for 36 h before coimmunoprecipitation and immunoblot analysis with the indicated antibodies. (D) DEFs were transfected with US3 or US3K213A plasmid; 24 h later, cells were either left untreated or transfected with ISD for 12 h before confocal microscopy. Bars, 10 μm.

Article Snippet: To confirm that DEV US3 mediates the phosphorylation of IRF7, cell lysates were subjected to a dephosphorylation assay using lambda protein phosphatase (PP) (New England Biolabs) ( ).

Techniques: Translocation Assay, Plasmid Preparation, Expressing, SDS Page, Western Blot, Transfection, Confocal Microscopy

Journal: Oncotarget

Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers

doi: 10.18632/oncotarget.10912

Figure Lengend Snippet: A. Western blot analysis of the expression levels of C16orf74 in pancreatic cancer cell lines. Control: Flag-tagged C16orf74-overexpressed diluted cell lysate. B. Phosphorylated form (arrow) of endogenous C16orf74 in KLM-1 cells, as examined by Western blot analysis using an anti-C16orf74 polyclonal antibody. The upper band disappeared when the cell lysate was incubated with lambda phosphatase (PPase (+)). C. Phosphorylation at threonine 44 (T44) of C16orf74. Flag-tagged wild type (WT), T41A and T44A mutants of C16orf74 were used to transfect COS-7 cells. The phosphorylated form of wild-type C16orf74 (arrow) was disappeared in the T44A mutant. D. Immunocytochemical analysis in a pancreatic cancer cell line (PK-1) using the anti-C16orf74 antibody, demonstrating the plasma membrane localization of endogenous C16orf74 (Green). DAPI staining is shown in blue.

Article Snippet: In the PPP3CA interaction assay, the KLM-1 cell lysate was incubated with lambda protein phosphatase, immunoprecipitated by mouse monoclonal PPP3CA (sc-17808, Santa Cruz) and immunoblotted with rabbit polyclonal C16orf74, as in the western blot analysis.

Techniques: Western Blot, Expressing, Control, Incubation, Phospho-proteomics, Mutagenesis, Clinical Proteomics, Membrane, Staining

Journal: Oncotarget

Article Title: Overexpression of C16orf74 is involved in aggressive pancreatic cancers

doi: 10.18632/oncotarget.10912

Figure Lengend Snippet: A. In vitro exogenous association of C16orf74 and PPP3CA. The Flag-tagged C16orf74 construct or vector alone was cotransfected with a myc-tagged PPP3CA construct into HEK293 cells. Cell lysates were immunoprecipitated using mouse anti-Flag antibody (left) or anti-myc antibody (right). Immunoblotting of the immunoprecipitates with rabbit anti-Flag or anti-myc antibodies revealed a specific interaction between the phosphorylated form of C16orf74 (arrow) and PPP3CA. B. In vitro endogenous association of C16orf74 and PPP3CA from Capan-1 pancreatic cancer cells, which endogenously express high levels of both C16orf74 and PPP3CA. Capan-1 cell lysates were immunoprecipitated using anti-C16orf74 antibody (left) or anti- PPP3CA antibody (right). Immunoblotting of the immunoprecipitates with anti-C16orf74 antibody or anti-PPP3CA antibodies revealed a specific interaction between C16orf74 and PPP3CA. Endogenous PPP3CA interacted with the phosphorylated form of endogenous C16orf74 (arrow). C. Interactions of wild-type C16orf74 (WT) and mutants of C16orf74 with PPP3CA, as assessed by IP analysis. Expression vectors for myc-His-tagged PPP3CA and Flag-tagged C16orf74 constructs were doubly transfected into HEK293T cells. C16orf74 (anti-Flag) was IP, and the indicated molecules were immunoblotted (IB) in western blot analysis. WT, replacement (T44A; non-phosphorylated form of C16orf74) and deletion mutants (∆PDIIIT; deletion mutant of PPP3CA binding motif) were analyzed. PPP3CA bound to wild-type C16orf74 but not the non-phosphorylated form of C16orf74 or the deletion mutant of the PPP3CA binding motif. D. Subcellular localization of C16orf74 (wild type or ∆PDIIIT) and PPP3CA in mammalian cells. Flag-tagged (green) C16orf74 (wild type or ∆PDIIIT) and myc-tagged (red) PPP3CA constructs were cotransfected into COS-7 cells and subjected to immunocytochemical staining. Flag-C16orf74 (wild type) and myc-PPP3CA colocalized on the under the cytoplasmic membrane of COS-7 cells (yellow), but Flag-C16orf74 (∆PDIIIT) did not colocalize with myc-PPP3CA, which was present diffusely in the cytoplasm. E. Interactions of endogenous C16orf74 with PPP3CA as assessed by IP analysis. The phosphorylated form (arrow) of endogenous C16orf74 in KLM-1 cells, as examined by western blot analysis using an anti-C16orf74 polyclonal antibody. Pre IP (left; non-immunoprecipitated by PPP3CA), the phosphorylated form of C16orf74 (upper band) disappeared when the cell lysate was incubated with lambda phosphatase (PPase (+)). Immunoprecipitation by PPP3CA (right) revealed that the phosphorylated form of C16orf74 (upper band) interacted with PPP3CA, whereas the non- phosphorylated form of C16orf74 did not. F. Invasion activity of wild-type C16orf74 (WT) and the two mutants (T44A: non-phosphorylated form of C16orf74; and ∆PDIIIT, deletion mutant of the PPP3CA binding motif). The WT-C16orf74 expression vector, T44A-C16orf74 expression vector, ∆PDIIIT-C16orf74 expression vector, and Mock vector were each transfected into NIH3T3 cells. The Matrigel invasion assay revealed an enhanced cell number for WT-C16orf74-over-expressing cells (3.4-fold, * P = 0.013) but not so enhanced for ∆PDIIIT-C16orf74-over-expressing cells (1.4-fold, ** P = 0.017) or T44A-C16orf74-over-expressing cells (2.3-fold,*** P = 0.038).

Article Snippet: In the PPP3CA interaction assay, the KLM-1 cell lysate was incubated with lambda protein phosphatase, immunoprecipitated by mouse monoclonal PPP3CA (sc-17808, Santa Cruz) and immunoblotted with rabbit polyclonal C16orf74, as in the western blot analysis.

Techniques: In Vitro, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing, Transfection, Mutagenesis, Binding Assay, Staining, Membrane, Incubation, Activity Assay, Invasion Assay

Journal: eLife

Article Title: Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit

doi: 10.7554/eLife.84694

Figure Lengend Snippet: ( A ) The wild-type and mutant condensin I complexes used in the current study. The recombinant complexes were purified from insect cells and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The gel was stained with Coomassie brilliant blue (CBB). ( B ) HaloTag–condensin I complexes used in the loop extrusion assay. The recombinant complexes were purified from insect cells and subjected to SDS–PAGE before being fluorescently labeled. The gel was stained with CBB. ( C ) Immunodepletion of endogenous condensins from Xenopus egg M-HSS. Endogenous condensin subunits were depleted from the M-HSS using Dynabeads Protein A coupled with control IgG (Δmock) or a mixture of antibodies against the subunits of condensins I and II (Δcond). To estimate the efficiency of depletion, the Δcond extract (100%) was compared with different amounts of the Δmock extract (5%, 20%, 50%, and 100%) by immunoblotting using the antibodies indicated. Endogenous topo IIα (XTopo IIα) was used as a loading control. The asterisk indicates a non-specific band. Figure 1—figure supplement 1—source data 1. Raw data uncropped gel corresponding to . Figure 1—figure supplement 1—source data 2. Microsoft excel of DNA constructs used in this study. Figure 1—figure supplement 1—source data 3. Raw data uncropped blots corresponding to .

Article Snippet: Recombinant DNA reagent , λDNA , New England Biolabs , N3011S , .

Techniques: Mutagenesis, Recombinant, Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Staining, Labeling, Immunodepletion, Control, Western Blot, Construct

Journal: eLife

Article Title: Cell cycle-specific loading of condensin I is regulated by the N-terminal tail of its kleisin subunit

doi: 10.7554/eLife.84694

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , λDNA , New England Biolabs , N3011S , .

Techniques: Plasmid Preparation, Recombinant, Purification, Sequencing, Mutagenesis, Cloning, Software, Western Blot, Immunodepletion, Immunoprecipitation

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Expression Plasmids Used in This Study

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Expressing, Plasmid Preparation, CRISPR, Sequencing, Construct, Luciferase

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Species specificity of mouse and human type III IFNs. (A) Table showing the relative antiviral activity of mouse and human IFN-λ on mouse LKR10 and human A549 cells. Species specificity index was calculated as the ratio between the relative antiviral activity on cells of homologous to nonhomologous species (eg, for mouse IFN: relative activity in mouse cells/relative activity in human cells). Relative activities in a given cell line were calculated as the IFN dilutions (starting from 1 ng/mL) that yielded similar antiviral activities. *Due to the low amount of IFN-λ4 available, the initial concentration of this IFN was estimated by comparison with human IFN-λ3 antiviral activity that was reported to have a similar specific activity. (B) Western blot showing STAT1 phosphorylation in mouse LKR10 cells 30 min after treatment with control medium (mock) or IFN-λ. Concentrations of human (Hu) IFN-λ3 (615 pg/mL) and IFN-λ4 that yielded the same antiviral activity on human A549 cells were used. As a control, mouse IFN-λ3 was used at a concentration (2.5 ng/mL) showing equivalent antiviral activity as human IFN-λ4 on mouse cells. Results are representative of 3 experiments.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Activity Assay, Concentration Assay, Comparison, Western Blot, Control

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: IFN-stimulated gene expression, STAT1 phosphorylation, and antiviral activity in response to IFN-αA and IFN-λ3 treatment in LKR10 derived cells. (A, B) Amounts of Oasl2 (A) and Usp18 (B) transcripts per 10 6 β-actin copies detected in WT and IFNAR2-KO LKR10 cells 24 h after treatment with mouse IFN-αA, IFN-λ3 (mIFN-λ3), or control medium (mock). (C) Percentage of mCherry-positive cells in WT and IFNAR2-KO LKR10 cells measured by flow cytometry 24 h postinfection with 0.5 PFU/cell TM967. Cells were pretreated with mouse IFN-αA, mIFN-λ3, or mock for 7 h before infection. (D) Western blot showing STAT1 phosphorylation and expression in LKR10 cells and derivatives. Cells were treated for 30 min before protein extraction. Reproducible results were obtained in 3 independent experiments. (A–D) 100 U/mL IFN-αA and 700 pg/mL mIFN-λ3 were used for treatment. Student's t -test: ***indicates a significant difference of the IFN-treated groups compared to the mock-treated group (A–C) . ns, nonsignificant. WT, wild-type.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Expressing, Activity Assay, Derivative Assay, Control, Flow Cytometry, Infection, Western Blot, Protein Extraction

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Mouse IFN-λ titration using Fawa-λ-luc reporter cells and ELISA. (A ) Luciferase activity detected in Fawa-λ-luc cells treated for the indicated time with 700 pg/mL mouse IFN-λ3 (mIFN-λ3). (B) Quantification by ELISA of mouse IFN-λ2 (mIFN-λ2) and mIFN-λ3 in cell supernatants. Two fold serial dilutions (800–12,800-fold) were quantified in quintuplicate. (C, D) Dose–response of mIFN-λ2 and mIFN-λ3 supernatants was measured in triplicate Fawa-λ-luc cells and is representative of at least 3 independent experiments. Cells were treated for 6 h with 2-fold serial dilutions. Data points in the linear range of the assays were plotted , and linear regression analysis was performed. LOD is based on the mean of mock treated cell signal, plus 3 standard deviations. ELISA, enzyme-linked immunosorbent assay; LOD, limit of detection; RLU, relative light units; R 2 , coefficient of regression.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Titration, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Human IFN-λ response in the mouse Fawa-λ-luc reporter cells. (A–D) Dose–response of human (hu) recombinant (A) IFN-λ1, (B) IFN-λ2, (C) IFN-λ3, and (D) IFN-λ4 as measured in triplicate. (A–D) Data points in the linear range of the assays were plotted , and linear regression analysis was performed. LOD is based on the mean of mock treated cell signal, plus 3 standard deviations.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Recombinant

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Sensitivity of IFN-λ to UV exposure and influence of serum on reporter cell activity and ELISA detection. (A, B) UV sensitivity of mouse and human IFN-λ. (A) Relative luciferase activities of Fawa-λ-luc cells treated with 250 pg/mL of mouse IFN-λ2 (mIFN-λ2) or IFN-λ3 (mIFN-λ3) supernatants irradiated ( n = 3) under 0.25, 0.5, 1 and 2 J/cm 2 . (B) Percentage of IFN-λ activity (mean and SD) remaining after UV treatment ( n = 3) at 2 J/cm 2 . IFN activity was measured in Fawa-λ-luc cells for IFN concentrations that yielded equivalent luciferase activities (20,000 RLU) before UV treatment (250 pg/mL mIFN-λ2 and mIFN-λ3, 125 pg/mL human IFN-λ1, 10 ng/mL huIFN-λ2, 500 pg/mL huIFN-λ3, and 62.5 pg/mL huIFN-λ4). (C, D) Influence of serum dilution on IFN-λ detection by Fawa-λ-luc cells (C) and ELISA (D) . (C) Fawa-λ-luc cells were treated in triplicate with fixed doses of 15 and 250 pg/mL mIFN-λ2 supernatant and with 2-fold serial dilutions (2–128-fold) of control mouse serum. (D) 125 pg/mL recombinant mIFN-λ3 was mixed with 2-fold serial dilutions of control mouse serum (2.5–10-fold) before detection by ELISA. (A–C) Reporter cells were exposed to IFN for 6 h before luciferase assay. Student's t -test: */**/***denotes a significant difference in signal compared to no UV exposure (A) or the absence of serum (C) . UV, ultraviolet light.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Irradiation, Control, Recombinant

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: Detection of type III IFN in biological samples by ELISA and bioassay. (A, B) IFN-λ2/3 detection by ELISA (A) and bioassay (B) in the serum of AG129 mice 2 or 4 days after electroinjection of mouse IFN-λ3 (mIFN-λ3) expressing (pCS59) or empty (pcDNA3) plasmids, 2 days after injection of pCS59, or 3 days after infection with mouse norovirus. IFN-λ detection in the serum by ELISA was performed without UV exposure to keep maximal sensitivity. (C, D) IFN-λ2/3 detection by ELISA (C) and bioassay (D) in the bronchoalveolar lavage of BALB/C mice, 5 days postinfection with RSV, compared to control mice (mock). BALF were UV-exposed before testing. (A–D) The horizontal dotted line represents the LOD. Mann–Whitney: */**indicates a significant difference compared to pcDNA3 group at days 2 or 4 (A, B) or compared to mock (D) . BALF, bronchoalveolar lavage fluid; RSV, respiratory syncytial virus.

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Enzyme-linked Immunosorbent Assay, Bioassay, Expressing, Injection, Infection, Control, MANN-WHITNEY, Virus

Journal: Journal of Interferon & Cytokine Research

Article Title: Species Specificity of Type III Interferon Activity and Development of a Sensitive Luciferase-Based Bioassay for Quantitation of Mouse Interferon-λ

doi: 10.1089/jir.2018.0066

Figure Lengend Snippet: IFN-λ sequence alignments. Sequences of human and mouse IFN-λ regions implicated in receptor binding were aligned. Key amino acid residues involved in receptor activation that differs between mouse and human IFN-λ2 and IFN-λ3 sequences are indicated in bold in mouse sequences. Residues unique to mouse IFN-λ3 in helices A and F are indicated in bold red . *Indicates identical amino acids between human IFN-λ3 and IFN-λ4. Color images available online at www.liebertpub.com/jir

Article Snippet: Recombinant mouse IFN-λ3 (1789-ML-025) and human IFN-λ1 (1598-IL-025), IFN-λ2 (1587-IL-025), and IFN-λ3 (5259-IL-025) were purchased from R&D (R&D Systems, Minneapolis).

Techniques: Sequencing, Binding Assay, Activation Assay

Journal: eLife

Article Title: Human RPA activates BLM’s bidirectional DNA unwinding from a nick

doi: 10.7554/eLife.54098

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Lambda DNA , Thermo Fisher Scientific , SD0021 , .

Techniques: Recombinant, Lambda DNA Preparation, Plasmid Preparation, Expressing, DNA Purification, Staining, Software

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Diagram of domains and motifs of human doublecortin-like kinase 1 (DCLK1) (UniProt O15075) that are conserved in the mouse DCLK1 used in this study. DC1, N-terminal doublecortin-like (DCX) domain; DC2, C-terminal DCX domain; kinase domain. Motifs enriched in PEST (proline/P, glutamic acid/E, serine/S, threonine/T) and DEND (aspartic acid/D, glutamic acid/E, asparagine/N, aspartic acid/D) based on and . Below: model of human DCLK1. DC1 domain (1mg4; ), DC2 domain modeled by homology to DCX-DC2 (5ip4; ), kinase domain (5jzj; ). DCLK1 is shown as a full-length pseudo-model, with projection domains/tails (and domain linkers) modeled as unfolded to visualize the length and convey the intrinsic disorder predicted for those regions. The mouse DCLK1 (1–740) used in this paper has the same amino acid boundaries as that of humans. ( B ) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Phos-tag gel of purified wild-type (WT), ΔC, and kinase-dead (D511N) DCLK1 proteins separated by phosphorylation level. Representative gel from n = 3 independent experiments. ( C ) Total internal reflection fluorescence microscopy (TIRF-M) images of 3 nM and 25 nM sfGFP-DCLK1 WT, ΔC, and D511N (green), expressed in bacteria under standard conditions, binding to taxol-stabilized microtubules (blue). Scale bars: 2.5 μm. ( D ) Quantification of microtubule-bound sfGFP-DCLK1 fluorescence intensity. Means ± sd: 2748.9 ± 2073.6 for 3 nM WT, 16119.6 ± 4324.3 for 25 nM WT, 1.2 ± 31.6 for 3 nM ΔC, 3.8 ± 101.9 for 25 nM ΔC, 9072.4 ± 3380.1 for 3 nM D511N, and 19666.6 ± 3345.3 for 25 nM D511N (n>100 microtubules from n = 3 independent trials for each concentration of each protein). Gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial. ***p<0.0001 and p = 0.3240 for 25 nM WT vs 25 nM D511N, calculated using Student’s t-test. p-values were calculated using n = 3. ( E ) Coomassie blue-stained SDS-PAGE Phos-tag gel of purified DCLK1-WT and -ΔC incubated with lambda phosphatase (λPP) or incubated in buffer alone for 1 hr at 30°C. Representative gel from n = 3 independent experiments. ( F ) TIRF-M images of 3 nM sfGFP-DCLK1 WT and ΔC (green) after treatment with λPP, binding to taxol-stabilized microtubules (blue). Scale bars: 2.5 μm. ( G ) Quantification of microtubule-bound sfGFP-DCLK1 fluorescence intensity. Means ± sd: 15090.7 ± 5285.6 for 3 nM WT + λPP, 18155.3 ± 3833.5 for 25 nM WT + λPP, 12004.2 ± 3490.3 for 3 nM ΔC + λPP, and 21240.6 ± 3413.5 for 25 nM ΔC + λPP (n>100 microtubules from n = 3 independent trials for each protein concentration; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial.). D511N data are reproduced from ( D ) for comparison. p = 0.3566 for 25 nM WT + λPP vs 25 nM ΔC + λPP, p = 0.6341 for 25 nM WT + λPP vs 25 nM D511N, p = 0.5989 for 25 nM ΔC + λPP vs 25 nM D511N, and p = 0.4462 for 3 nM WT + λPP vs 3 nM ΔC + λPP, calculated using Student’s t-test. p-values were calculated using n = 3. For all experiments, at least two separate protein purifications were used. Figure 1—source data 1. Uncropped gels for the associated panels in . The red box indicates how the gel was cropped. Figure 1—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Phospho-proteomics, Fluorescence, Microscopy, Bacteria, Binding Assay, Concentration Assay, Incubation, Protein Concentration, Comparison

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of sfGFP-DCLK1 proteins used in this study. ( B ) Anion exchange elution chromatograms for wild-type doublecortin-like kinase 1 (DCLK1-WT) compared to -T687A, -T688A, and -D511N (left) and DCLK1-ΔC compared to ∆C DC1-4A and ∆C DC2-4A (right), showing that all proteins elute in a homogenous peak at approximately the same ionic strength, revealing that they are similarly folded. ( C ) Anion exchange elution chromatograms for DCLK1-WT and DCLK1-ΔC prepped in the presence or absence of lambda phosphatase (λPP). The non-phosphorylated proteins (+λPP) elute at a lower ionic strength consistent with a lower net negative charge. Note the larger shift in the elution volume for the non-phosphorylated vs phosphorylated DCLK1-ΔC, indicating a more pronounced change in the net charge between the two preps. Figure 1—figure supplement 1—source data 1. Uncropped gels for the associated panels in . The red box indicates how the gel was cropped. Figure 1—figure supplement 1—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Staining, Polyacrylamide Gel Electrophoresis, SDS Page

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Immunoblots of strepII-sfGFP-tagged wild-type (WT) or kinase-dead (D511N) doublecortin-like kinase 1 (DCLK1) incubated in the absence or presence of ATPγS for 30 min at 37°C. DCLK1-WT, but not DCLK1-D511N, robustly autophosphorylates. ( B ) Immunoblots of strepII-sfGFP-tagged DCLK1-D511N incubated with an untagged version of DCLK1-WT to distinguish the proteins by size in the absence or presence of ATPγS for 30 min at 37°C. While DCLK1-WT autophosphorylated, there was still no detectable level of phosphorylation for DCLK1-D511N. For ( A ) and ( B ), primary antibodies used were mouse anti-strep (Fisher NBP243719) and rabbit anti-thiophosphate ester (Abcam ab133473). For both ( A ) and ( B ), these blots are representative images from at least n = 3 independent experiments. ( C ) Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Phos-tag gel of purified DCLK1-WT, -ΔC, and -D511N incubated with lambda phosphatase (λPP) or incubated in buffer alone for 1 hr at 30°C. Representative gel from n = 3 independent experiments. ( D ) Representative total internal reflection fluorescence microscopy (TIRF-M) image of 10 nM sfGFP-DCLK1-ΔC (green, co-expressed in bacteria with λPP) binding to taxol-stabilized microtubules (blue) after a 30-min incubation in the presence of 2 mM adenosine triphosphate (ATP). Scale bar: 5 μm. ( E ) Coomassie blue-stained SDS-PAGE shows the binding behavior of 500 nM DCLK1-WT or -ΔC in the absence or presence of 2 mM ATP in the absence or presence of 2 μM taxol-stabilized microtubules. In the absence of ATP, the percent (means ± sd) of DCLK1 that co-pelleted with microtubules was 99.3 ± 0.5% for WT and 96.3 ± 1.4% for ΔC (n = 3 independent experiments; p = 0.0250). In the presence of ATP, the percent (means ± sd) of DCLK1 that co-pelleted with microtubules was 86.2 ± 4.9% for WT and 6.9 ± 5.0% for ΔC (n = 3 independent experiments; p<0.0001). For all experiments, at least two separate protein purifications were used. Figure 1—figure supplement 2—source data 1. Uncropped blots ( A, B ), gels ( C, E ) for the associated panels in . The red box indicates how the blot of gel was cropped. Figure 1—figure supplement 2—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Western Blot, Incubation, Phospho-proteomics, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Fluorescence, Microscopy, Bacteria, Binding Assay

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Coomassie blue-stained sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) Phos-tag gel of purified doublecortin-like kinase 1 wild-type (DCLK1-WT) and -ΔC proteins separated by phosphorylation level. The first and fifth lanes contain DCLK1-WT and -ΔC expressed in bacteria under standard conditions. All other lanes contain DCLK1-WT and -ΔC that were co-expressed with lambda phosphatase (λPP), which was subsequently separated from DCLK1. Incubation of λPP-treated DCLK1-WT and -ΔC with 2 mM adenosine triphosphate (ATP) at the indicated times reveals a band shift, indicative of an increase in phosphorylation. ( B ) Quantification of the average percent of total DCLK1 protein that is phosphorylated in each condition. Averages are derived from n = 3 independent experiments. ( C ) Total internal reflection fluorescence microscopy (TIRF-M) images of sfGFP-DCLK1-WT and -ΔC (co-expressed in bacteria with λPP) at indicated concentrations (green) binding to taxol-stabilized microtubules (blue) after a 30-min incubation in the absence or presence of 2 mM ATP. Scale bars: 2.5 μm. ( D ) Quantification of microtubule-bound sfGFP-DCLK1-WT fluorescence intensity plotted against concentration after a 30-min incubation in the absence or presence of ATP (WT without ATP, K D = 2.1 nM, and WT with ATP, K D = 5.4 nM, derived from at least n = 3 independent trials per condition). ( E ) Quantification of microtubule-bound sfGFP-DCLK1-ΔC fluorescence intensity plotted against concentration after a 30-min incubation in the absence or presence of ATP (ΔC without ATP, K D = 3.9 nM, and ΔC with ATP, K D = 161.0 nM, derived from n = 3 independent trials). ( F ) TIRF-M images of 10 nM sfGFP-DCLK1-WT or -ΔC (green, co-expressed in bacteria with λPP) binding to non-stabilized GDP microtubules grown from GMPCPP seeds (blue) after a 30-min incubation in the absence or presence of 2 mM ATP. Scale bars: 2.5 μm. ( G ) Quantification of microtubule-bound sfGFP-DCLK1 fluorescence intensity. Means ± sd: 15004.4 ± 6503.8 for WT, 12535.9 ± 3247.5 for WT + ATP, 12111.3 ± 3534.0 for ΔC, and 1579.3 ± 866.5 for ΔC + ATP (n>60 microtubules from n = 2 independent trials for each condition; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.6360 for WT vs ΔC and p = 0.0440 for WT + ATP vs ΔC + ATP, calculated using Student’s t-test; p-values were calculated using n = 2). For all experiments, at least two separate protein purifications were used. Figure 2—source data 1. Uncropped gel for the associated panel in . The red box indicates how the gel was cropped. Figure 2—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Phospho-proteomics, Bacteria, Incubation, Electrophoretic Mobility Shift Assay, Derivative Assay, Fluorescence, Microscopy, Binding Assay, Concentration Assay

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Diagrams depicting the domains, amino acid boundaries, and mutations relevant to the doublecortin-like kinase 1 (DCLK1) constructs used. ∆C DC1-4A indicates the four residues in DC1 that were mutated to alanines S77, S83, S96, and T143. ∆C DC2-4A indicates the four residues in DC2 that were mutated to alanines T189, S193, T218, and S228. ( B ) Total internal reflection fluorescence microscopy (TIRF-M) images of sfGFP-DCLK1 ΔC, ∆C DC1-4A , and ∆C DC2-4A , co-expressed in bacteria with lambda phosphatase (λPP), at indicated concentrations binding to taxol-stabilized microtubules (blue) in the absence or presence of adenosine triphosphate (ATP). Scale bars: 2.5 μm. ( C ) Quantification of microtubule-bound 5 nM sfGFP-DCLK1 fluorescence intensity. For 5 nM concentrations in the absence of ATP, means ± sd: 12883.5 ± 2881.6 for ΔC, 12245.5 ± 3283.0 for ∆C DC1-4A , 8552.7 ± 2097.3 for ∆C DC2-4A (n>100 microtubules per condition from n = 3 independent trials; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.8128 for ΔC vs ∆C DC1-4A and p = 0.1031 for ΔC vs ∆C DC2-4A calculated using Student’s t-test; p-values were calculated using n = 3). For 5 nM concentrations in the presence of ATP, means ± sd: 987.6 ± 202.5 for ΔC + ATP, 4042.6 ± 1624.6 for ∆C DC1-4A + ATP, 2482.0 ± 1058.3 for ∆C DC2-4A + ATP (n>100 microtubules from n = 3 independent trials; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.0319 for ΔC vs ∆C DC1-4A and p = 0.0742 for ΔC vs ∆C DC2-4A calculated using Student’s t-test; p-values were calculated using n = 3). ( D ) Quantification of microtubule-bound 20 nM sfGFP-DCLK1 fluorescence intensity. For 20 nM concentrations in the absence of ATP, means ± sd: 23634.3 ± 1725.1 for ΔC, 22277.3 ± 1334.9 for ∆C DC1-4A , 19912.2 ± 5408.6 for ∆C DC2-4A (n>100 microtubules per condition from n = 3 independent trials; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.3419 for ΔC vs ∆C DC1-4A and p = 0.3196 for ΔC vs ∆C DC2-4A calculated using Student’s t-test; p-values were calculated using n = 3). For 20 nM concentrations in the presence of ATP, means ± sd: 1579.8 ± 585.1 for ΔC + ATP, 12556.9 ± 3419.9 for ∆C DC1-4A + ATP, 3503.4 ± 826.7 for ∆C DC2-4A + ATP (n>100 microtubules from n = 4, 6, and 5 independent trials for ΔC, ∆C DC1-4A , and ∆C DC2-4A , respectively; gray dots indicate individual microtubule intensities, while colored dots represent the averages from each trial; p = 0.0002 for ΔC vs ∆C DC1-4A and p = 0.0058 for ΔC vs ∆C DC2-4A calculated using Student’s t-test; p-values were calculated using n = number of independent trials as stated above). For all experiments, at least two separate protein purifications were used.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Construct, Fluorescence, Microscopy, Bacteria, Binding Assay

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Diagram depicting the domains, amino acid boundaries, and mutations in the C-terminal region relevant to the doublecortin-like kinase 1 (DCLK1) constructs used. ( B ) Coomassie blue-stained sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) Phos-tag gel of purified kinase-dead (D511N), WT, T687A, and T688A DCLK1 proteins separated by phosphorylation level. For all experiments, DCLK1 proteins were co-expressed with lambda phosphatase (λPP), which was subsequently separated from DCLK1. Incubation of λPP-treated DCLK1 proteins with 2 mM adenosine triphosphate (ATP) at the indicated times reveals a shift in the phosphorylation level to varying degrees. ( C ) Quantification of the average percent of total DCLK1 protein that is phosphorylated in each condition. Averages were derived from n = 3 independent experiments. ( D ) Total internal reflection fluorescence microscopy (TIRF-M) images of sfGFP-DCLK1-WT, -T687A, and -T688A (co-expressed in bacteria with λPP) at indicated concentrations (green) binding to taxol-stabilized microtubules (blue) after a 30-min incubation in the absence or presence of 2 mM ATP. Scale bars: 2.5 μm. ( E ) Quantification of microtubule-bound sfGFP-DCLK1-WT, -T687A, and -T688A fluorescence intensity plotted against concentration after a 30-min incubation in the absence of ATP (K D = 3.0 nM, 2.8 nM, and 2.6 nM for WT, T687A, and T688A, respectively, from at least n = 3 independent trials per condition). ( F ) Quantification of microtubule-bound sfGFP-DCLK1-WT, -T687A, and -T688A fluorescence intensity plotted against concentration after a 30-min incubation with ATP (K D = 5.7 nM, 3.9 nM, and 239.2 nM for WT, T687A, and T688A, respectively, from at least n = 3 independent trials per condition). ( G ) Coomassie blue-stained SDS-PAGE shows the binding behavior of 500 nM DCLK1-WT or -T688A in the absence or presence of 2 mM ATP in the absence or presence of 2 μM taxol-stabilized microtubules. In the absence of ATP, the percent (means ± sd) of DCLK1 that co-pelleted with microtubules was 99.3 ± 0.5% for WT and 98.9 ± 1.0% for T688A (n = 3 independent experiments; p = 0.5690). In the presence of ATP, the percent (means ± sd) of DCLK1 that co-pelleted with microtubules was 86.2 ± 4.9% for WT and 7.9 ± 3.5% for T688A (n = 3 independent experiments; p<0.0001). For all experiments, at least two separate protein purifications were used. Figure 5—source data 1. Uncropped gels for the associated panels in . The red box indicates how the gel was cropped. Figure 5—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Construct, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Phospho-proteomics, Incubation, Derivative Assay, Fluorescence, Microscopy, Bacteria, Binding Assay, Concentration Assay

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet: ( A ) Coomassie blue-stained sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) gels of purified doublecortin-like kinase 1 (DCLK1) proteins fractionated by sucrose density-gradient centrifugation. Representative gels from n = 2 independent experiments for each protein are shown. ( B ) Quantification of the average percent of DCLK1 protein in each fraction, revealing a peak in fraction 4 for all DCLK1 proteins. ( C ) Coomassie blue-stained SDS-PAGE gel of purified DCLK1-WT and -T688A proteins co-expressed with lambda phosphatase (λPP) in bacteria, which was first incubated either in the absence or in the presence of adenosine triphosphate (ATP) for 30 min at 25°C followed by an incubation with calpain for 15 min at 30°C. Similar band patterns are seen for each protein under each condition after cleavage with calpain. Gel is representative of n = 3 independent experiments. Figure 5—figure supplement 1—source data 1. Uncropped gels for the associated panels in . The red box indicates how the gel was cropped. Figure 5—figure supplement 1—source data 2. Uncropped gels.

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Gradient Centrifugation, Bacteria, Incubation

Journal: eLife

Article Title: Autoregulatory control of microtubule binding in doublecortin-like kinase 1

doi: 10.7554/eLife.60126

Figure Lengend Snippet:

Article Snippet: The cDNAs (complementary DNA) used for protein expression in this study were as follows: mouse DCLK1 (Transomic, BC133685) and Lambda phosphatase (Addgene, 79748, RRID: Addgene_79748 ).

Techniques: Microscopy, Recombinant, Software

Journal: Cell

Article Title: Non-Coding Transcription Instructs Cohesin-Dependent Chromatin Folding and Compartmentalization to Dictate Enhancer-Promoter Communication and T Cell Fate

doi: 10.1016/j.cell.2017.09.001

Figure Lengend Snippet: TABLE FOR AUTHOR TO COMPLETE

Article Snippet: Anti-Lamin B1 , Santa Cruz , Cat # sc-6217 RRID:AB_648158.

Techniques: Recombinant, Lambda DNA Preparation, Protease Inhibitor, Ligation, Electron Microscopy, Sample Prep, Multiplex Assay, Membrane, Nick Translation, Plasmid Preparation, Clone Assay, Sequencing, Control, Software, Microscopy