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  • 99
    Thermo Fisher sds page gradient gels
    Sds Page Gradient Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 458 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis
    Gradient Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad gradient sds page gels
    Interaction between eEF1A and SidI truncations in the absence of SusF. Lysates from E. coli expressing GST-SidI constructs were incubated with glutathione agarose beads followed by washing and incubation with lysates from HEK 293 cells. Proteins were separated by <t>SDS-PAGE</t> and visualized by Coomassie staining (GST-SidI) or Western blotting (eEF1A). Data are representative of two independent experiments.
    Gradient Sds Page Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 1437 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gradient sds page gels
    DipA associates with PxdA-marked endosomes. (A) Sypro Ruby-stained <t>SDS-PAGE</t> gel of lysates incubated with HA-conjugated agarose from wild-type (Ctrl) or PxdA-HA expressing hyphae. Mass spectrometry identified DipA (indicated by arrow) from two biological replicates. (B) Representative kymograph of DipA-GFP movement. Also see Video 3. (C) Histogram of DipA-GFP velocities calculated from kymographs as in (B). Mean velocities are 2.44 ± 0.77 (SD) μm/sec. n = 257 moving events. (D) Colocalization of DipA-GFP and PxdA-mKate along a hypha. (E) Representative kymograph of DipA and PxdA co-movement. (F) Quantification of the percent overlap of DipA colocalized with PxdA. Mean percent overlap is 97.88 ± 6.01 (SD). N = 8 kymographs. (G) Representative image of DipA-GFP distribution in hookAΔ hyphae. (H) DipA-GFP distribution is quantified by fluorescence intensity line-scans of fluorescently-tagged organelles in wild-type (WT) or hookAΔ hyphae. Mean fluorescence intensity (solid lines) ± SEM (shading) is plotted as a function of distance from the hyphal tip. n = 3 (WT) and 7 ( hookAΔ ) hyphae.
    Gradient Sds Page Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 664 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sds page gradient gels
    IFNγ-dependent engagement of mTOR effectors is Sin1-dependent. A–I , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. A–I ( top panels ), cell lysates were prepared, and equal amounts of protein were resolved by <t>SDS-PAGE</t> and then subjected to immunoblotting with the indicated specific anti-phosphoantibodies. The blots in the respective top panels were stripped and probed with anti-AKT ( A–C ), anti-mTOR ( D and E ), anti-4E-BP1 ( F ), anti-p70S6K ( G ), anti-rpS6 ( H ), and anti-eIF4B ( I ) antibodies. J–N , serum-starved U937 cells transfected with control siRNA or Sin1 siRNA were treated with human IFNγ (5 × 10 3 IU/ml) for the indicated times. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and then processed for immunoblotting with anti- phospho-Ser-473 AKT, AKT, Sin1, and GAPDH antibodies ( J ), anti-phospho-Thr-308 AKT and anti-AKT antibodies ( K ), antibodies against phospho-Ser-2481 mTOR, mTOR, Sin1, or GAPDH ( L ), anti-phospho-Thr-37/46 4E-BP1 and anti-4E-BP1 antibodies ( M ), and anti-phospho-Thr-389 p70S6K and anti-p70S6K antibodies ( N ). A–K ( bottom panels ), bands from three independent experiments (including the blots shown) were quantified by densitometry. Data are expressed as ratios of phosphoprotein over respective total protein values, and bar graphs represent means ± S.E. ( error bars ) of three independent experiments for each experimental condition.
    Sds Page Gradient Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 564 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad gradient sds polyacrylamide gel electrophoresis
    IFNγ-dependent engagement of mTOR effectors is Sin1-dependent. A–I , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. A–I ( top panels ), cell lysates were prepared, and equal amounts of protein were resolved by <t>SDS-PAGE</t> and then subjected to immunoblotting with the indicated specific anti-phosphoantibodies. The blots in the respective top panels were stripped and probed with anti-AKT ( A–C ), anti-mTOR ( D and E ), anti-4E-BP1 ( F ), anti-p70S6K ( G ), anti-rpS6 ( H ), and anti-eIF4B ( I ) antibodies. J–N , serum-starved U937 cells transfected with control siRNA or Sin1 siRNA were treated with human IFNγ (5 × 10 3 IU/ml) for the indicated times. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and then processed for immunoblotting with anti- phospho-Ser-473 AKT, AKT, Sin1, and GAPDH antibodies ( J ), anti-phospho-Thr-308 AKT and anti-AKT antibodies ( K ), antibodies against phospho-Ser-2481 mTOR, mTOR, Sin1, or GAPDH ( L ), anti-phospho-Thr-37/46 4E-BP1 and anti-4E-BP1 antibodies ( M ), and anti-phospho-Thr-389 p70S6K and anti-p70S6K antibodies ( N ). A–K ( bottom panels ), bands from three independent experiments (including the blots shown) were quantified by densitometry. Data are expressed as ratios of phosphoprotein over respective total protein values, and bar graphs represent means ± S.E. ( error bars ) of three independent experiments for each experimental condition.
    Gradient Sds Polyacrylamide Gel Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gradient sds polyacrylamide gel electrophoresis
    IFNγ-dependent engagement of mTOR effectors is Sin1-dependent. A–I , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. A–I ( top panels ), cell lysates were prepared, and equal amounts of protein were resolved by <t>SDS-PAGE</t> and then subjected to immunoblotting with the indicated specific anti-phosphoantibodies. The blots in the respective top panels were stripped and probed with anti-AKT ( A–C ), anti-mTOR ( D and E ), anti-4E-BP1 ( F ), anti-p70S6K ( G ), anti-rpS6 ( H ), and anti-eIF4B ( I ) antibodies. J–N , serum-starved U937 cells transfected with control siRNA or Sin1 siRNA were treated with human IFNγ (5 × 10 3 IU/ml) for the indicated times. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and then processed for immunoblotting with anti- phospho-Ser-473 AKT, AKT, Sin1, and GAPDH antibodies ( J ), anti-phospho-Thr-308 AKT and anti-AKT antibodies ( K ), antibodies against phospho-Ser-2481 mTOR, mTOR, Sin1, or GAPDH ( L ), anti-phospho-Thr-37/46 4E-BP1 and anti-4E-BP1 antibodies ( M ), and anti-phospho-Thr-389 p70S6K and anti-p70S6K antibodies ( N ). A–K ( bottom panels ), bands from three independent experiments (including the blots shown) were quantified by densitometry. Data are expressed as ratios of phosphoprotein over respective total protein values, and bar graphs represent means ± S.E. ( error bars ) of three independent experiments for each experimental condition.
    Gradient Sds Polyacrylamide Gel Electrophoresis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher bis tris sds page gradient gels
    Restricting CDKL5 expression to the cell nucleus. ( A ) Extracts of CDKL5 disrupted U–2–OS (Flp-In T-Rex) cells ( CDKL5 Δ/Δ ) stably expressing CDKL5 NLS –WT or a K 42 R kinase-dead mutant (CDKL5 NLS –KD) or empty vector were subjected to western blotting with the antibodies indicated. Two different dishes of cells are shown per condition. ( B ) CDKL5 Δ/Δ cells stably expressing CDKL5, CDKL5 NLS –WT or CDKL5 NLS –KD were subjected to indirect immunofluorescence analysis with anti-CDKL5 antibodies. ( C ) Subcellular fractionation of lysates from CDKL5 ΄/Δ cells stably expressing CDKL5, CDKL5 NLS –WT or CDKL5 NLS –KD or empty vector. Lysates were fractionated to isolate proteins found in the following subcellular compartments: cytoplasmic (Cyt), membrane (Mb), nuclear (Nuc), chromatin (Chr) or cytoskeleton (Csk). Fractionated samples were resolved by <t>SDS-PAGE</t> and probed with antibodies shown. ( D ) CDKL5 Δ/Δ cells stably expressing CDKL5 NLS –WT or CDKL5 NLS –KD (or empty vector) were treated with 500 μM H 2 O 2 for 15 min. Samples were resolved by SDS-PAGE and probed with indicated antibodies or stained with Ponceau S to show equal loading. Rep=biological replicate.
    Bis Tris Sds Page Gradient Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis
    Restricting CDKL5 expression to the cell nucleus. ( A ) Extracts of CDKL5 disrupted U–2–OS (Flp-In T-Rex) cells ( CDKL5 Δ/Δ ) stably expressing CDKL5 NLS –WT or a K 42 R kinase-dead mutant (CDKL5 NLS –KD) or empty vector were subjected to western blotting with the antibodies indicated. Two different dishes of cells are shown per condition. ( B ) CDKL5 Δ/Δ cells stably expressing CDKL5, CDKL5 NLS –WT or CDKL5 NLS –KD were subjected to indirect immunofluorescence analysis with anti-CDKL5 antibodies. ( C ) Subcellular fractionation of lysates from CDKL5 ΄/Δ cells stably expressing CDKL5, CDKL5 NLS –WT or CDKL5 NLS –KD or empty vector. Lysates were fractionated to isolate proteins found in the following subcellular compartments: cytoplasmic (Cyt), membrane (Mb), nuclear (Nuc), chromatin (Chr) or cytoskeleton (Csk). Fractionated samples were resolved by <t>SDS-PAGE</t> and probed with antibodies shown. ( D ) CDKL5 Δ/Δ cells stably expressing CDKL5 NLS –WT or CDKL5 NLS –KD (or empty vector) were treated with 500 μM H 2 O 2 for 15 min. Samples were resolved by SDS-PAGE and probed with indicated antibodies or stained with Ponceau S to show equal loading. Rep=biological replicate.
    Gradient Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher tris glycine sds page gradient gels
    Dynamin GTPases are modestly phosphorylated by LRRK2 in vitro. In vitro kinase assays with [ 33 P]-γ-ATP, soluble recombinant full-length FLAG-tagged LRRK2 variants (WT, G2019S or D1994A) and immunopurified ‘on-bead’ YFP-tagged ArfGAP1 ( A ), GFP-tagged Dnm1 ( B ), and Myc-tagged Drp1 ( C ), Mfn1 ( D ), Mfn2 ( E ) or OPA1 ( F ), derived by IP from transfected HEK-293T cells. Following kinase reactions, soluble LRRK2 and ‘on-bead’ substrates were separated and resolved on independent <t>SDS–PAGE</t> gels, as indicated. Western blot analysis with anti-GFP, anti-myc or anti-FLAG antibodies indicate equal loading of ArfGAP1, Dnm1, Drp1, Mfn1, Mfn2, OPA1 and LRRK2 proteins in each condition. Autoradiographs ( 33 P) reveal the LRRK2-dependent phosphorylation of ArfGAP1, Dnm1, Drp1, Mfn1 and OPA1, with enhanced phosphorylation by G2019S LRRK2 compared with WT or kinase-inactive D1994A LRRK2. A soluble eluate from FLAG IPs (derived from non-transfected cells) was used as a control in each assay to assess background 33 P incorporation for each substrate. LRRK2 autophosphorylation is also detected in these assays. Blots are representative of at least three-independent kinase experiments.
    Tris Glycine Sds Page Gradient Gels, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad gradient tris glycine sds page gel
    Dynamin GTPases are modestly phosphorylated by LRRK2 in vitro. In vitro kinase assays with [ 33 P]-γ-ATP, soluble recombinant full-length FLAG-tagged LRRK2 variants (WT, G2019S or D1994A) and immunopurified ‘on-bead’ YFP-tagged ArfGAP1 ( A ), GFP-tagged Dnm1 ( B ), and Myc-tagged Drp1 ( C ), Mfn1 ( D ), Mfn2 ( E ) or OPA1 ( F ), derived by IP from transfected HEK-293T cells. Following kinase reactions, soluble LRRK2 and ‘on-bead’ substrates were separated and resolved on independent <t>SDS–PAGE</t> gels, as indicated. Western blot analysis with anti-GFP, anti-myc or anti-FLAG antibodies indicate equal loading of ArfGAP1, Dnm1, Drp1, Mfn1, Mfn2, OPA1 and LRRK2 proteins in each condition. Autoradiographs ( 33 P) reveal the LRRK2-dependent phosphorylation of ArfGAP1, Dnm1, Drp1, Mfn1 and OPA1, with enhanced phosphorylation by G2019S LRRK2 compared with WT or kinase-inactive D1994A LRRK2. A soluble eluate from FLAG IPs (derived from non-transfected cells) was used as a control in each assay to assess background 33 P incorporation for each substrate. LRRK2 autophosphorylation is also detected in these assays. Blots are representative of at least three-independent kinase experiments.
    Gradient Tris Glycine Sds Page Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenScript gradient sds page gels
    <t>SDS–PAGE</t> and a ligand blot analysis of the HaABCC1 fragments. (A) Marker, protein marker; Lane 1, TMD1 fragment after ultrasound treatment; Lane 2, TMD2 fragment after ultrasound treatment. (B) Lanes 1 and 4, protein marker; Lanes 3 and 6, bovine serum albumin (BSA), as a control; Lane 2, TMD1 fragment bound with Cry2Ab; Lane 5 TMD2 fragment bound with Cry2Ab.
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    Thermo Fisher bis tris sds page gradient gel
    Sindbis virus (SINV) virion purification strategy and production flowchart. (A) Clarified viral preparations were purified over two potassium tartrate density gradients done in series using isopycnic ultracentrifugation and a final wash and pellet step. Negative controls (uninfected cell culture supernatant) were processed in parallel with the virus preparations. (B) Transmission electron micrographs were taken of the purified virions to confirm efficiency of this enrichment procedure. (C) An <t>SDS-PAGE</t> gel was stained with silver or with Coomassie blue for each of the viral preparations. HepG2 preparations are presented here and are representative of all viral preparations from all host cellular backgrounds.
    Bis Tris Sds Page Gradient Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    FUJIFILM gradient sds page gels
    Subcellular fractionation of mouse cerebral cortex . (A) Schematic representation of the subcellular fractionation steps. P1, nuclear pellet and debris; P2, crude synaptosomal fraction; P3, light membranes; S3, cytosolic fraction; LP1, synaptosomal membrane fraction; LP2, synaptic vesicle-enriched fraction; LS2, soluble synaptosomal fraction. (B) Western blots of JNPL3 and non-tg male mouse cerebral cortex subcellular fractions. P1, P2, P3, or S3 fraction derived from 0.13 mg wet weight of tissue and LP1, LP2, or LS2 fraction derived from 0.5 mg wet weight of tissue were loaded on <t>SDS-PAGE.</t> Blots were probed with PSD-95, synaptotagmin, GAP-43, Tau5, E1, β-tubulin, and β-actin antibodies. (C) Proportions of protein levels in fractions (P1, P2, P3, and S3) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice are shown. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (P1 + P2 + P3 + S3). Results are expressed as mean ± SEM. (D) Proportions of protein levels in synaptosomal fractions (LP1, LP2, and LS2) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice were indicated. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (LP1 + LP2 + LS2). Results are expressed as mean ± SEM.
    Gradient Sds Page Gels, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sds page gradient gel electrophoresis
    Subcellular fractionation of mouse cerebral cortex . (A) Schematic representation of the subcellular fractionation steps. P1, nuclear pellet and debris; P2, crude synaptosomal fraction; P3, light membranes; S3, cytosolic fraction; LP1, synaptosomal membrane fraction; LP2, synaptic vesicle-enriched fraction; LS2, soluble synaptosomal fraction. (B) Western blots of JNPL3 and non-tg male mouse cerebral cortex subcellular fractions. P1, P2, P3, or S3 fraction derived from 0.13 mg wet weight of tissue and LP1, LP2, or LS2 fraction derived from 0.5 mg wet weight of tissue were loaded on <t>SDS-PAGE.</t> Blots were probed with PSD-95, synaptotagmin, GAP-43, Tau5, E1, β-tubulin, and β-actin antibodies. (C) Proportions of protein levels in fractions (P1, P2, P3, and S3) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice are shown. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (P1 + P2 + P3 + S3). Results are expressed as mean ± SEM. (D) Proportions of protein levels in synaptosomal fractions (LP1, LP2, and LS2) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice were indicated. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (LP1 + LP2 + LS2). Results are expressed as mean ± SEM.
    Sds Page Gradient Gel Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    NuSep sds page gradient gels
    GelE cleavage of rAce analyzed by <t>SDS-PAGE.</t> Sample lanes from left to right represent reaction mixtures containing 30 μg of rAce/ml with various amounts of purified GelE (0.015, 0.045, 0.15, 0.45. 1.5, 4.5, and 15 μg/ml), rAce (30 μg/ml)
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    Image Search Results


    Interaction between eEF1A and SidI truncations in the absence of SusF. Lysates from E. coli expressing GST-SidI constructs were incubated with glutathione agarose beads followed by washing and incubation with lysates from HEK 293 cells. Proteins were separated by SDS-PAGE and visualized by Coomassie staining (GST-SidI) or Western blotting (eEF1A). Data are representative of two independent experiments.

    Journal: bioRxiv

    Article Title: The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

    doi: 10.1101/845313

    Figure Lengend Snippet: Interaction between eEF1A and SidI truncations in the absence of SusF. Lysates from E. coli expressing GST-SidI constructs were incubated with glutathione agarose beads followed by washing and incubation with lysates from HEK 293 cells. Proteins were separated by SDS-PAGE and visualized by Coomassie staining (GST-SidI) or Western blotting (eEF1A). Data are representative of two independent experiments.

    Article Snippet: SDS-PAGE and Western Blot Boiled protein samples were loaded onto either 4-20% gradient SDS-PAGE gels (BioRad), 12% or 15% SDS-PAGE gels.

    Techniques: Expressing, Construct, Incubation, SDS Page, Staining, Western Blot

    Co-elution of SusF and SidI by gel filtration chromatography. A sample of recombinant SidI bound to SusF was separated by analytical scale gel filtration chromatography as presented in Figure 1C . Ten µl of each column fraction were analyzed by SDS-PAGE followed by Coomassie staining to assess the protein content within each fraction. Fractions corresponding to the peak of approximately 150 kDa apparent molecular weight in the sample of SidI bound to SusT ( Fig. 1C , red trace) contained both proteins, consistent with formation of a 1:1 complex between these two molecules.

    Journal: bioRxiv

    Article Title: The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

    doi: 10.1101/845313

    Figure Lengend Snippet: Co-elution of SusF and SidI by gel filtration chromatography. A sample of recombinant SidI bound to SusF was separated by analytical scale gel filtration chromatography as presented in Figure 1C . Ten µl of each column fraction were analyzed by SDS-PAGE followed by Coomassie staining to assess the protein content within each fraction. Fractions corresponding to the peak of approximately 150 kDa apparent molecular weight in the sample of SidI bound to SusT ( Fig. 1C , red trace) contained both proteins, consistent with formation of a 1:1 complex between these two molecules.

    Article Snippet: SDS-PAGE and Western Blot Boiled protein samples were loaded onto either 4-20% gradient SDS-PAGE gels (BioRad), 12% or 15% SDS-PAGE gels.

    Techniques: Co-Elution Assay, Filtration, Chromatography, Recombinant, SDS Page, Staining, Molecular Weight

    SusF does not impair interaction between SidI and eEF1A. (A) GST-SusF or GST alone were immobilized on magnetic glutathione agarose beads followed by incubation with 100 µg purified His6-SidI alone or that had been preincubated with lysates from HEK 293T cells as indicated (Lysates). Proteins were separated by SDS-PAGE and visualized by Coomassie staining or Western blot as indicated. (B) Lysates from E. coli expressing GST-SidI or GST alone were incubated with magnetic glutathione agarose beads and lysates from HEK 293T followed by 10, to 1000 µg of purified recombinant SusF (shown as increasing amounts in supernatants from beads). Proteins remaining on the beads were separated by SDS-PAGE and visualized by Coomassie stain or Western blot as indicated. GST-SidI (∼130 kDa) and SusF (∼27 kDa) are indicated with arrowheads. Data are representative of at least two independent experiments.

    Journal: bioRxiv

    Article Title: The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

    doi: 10.1101/845313

    Figure Lengend Snippet: SusF does not impair interaction between SidI and eEF1A. (A) GST-SusF or GST alone were immobilized on magnetic glutathione agarose beads followed by incubation with 100 µg purified His6-SidI alone or that had been preincubated with lysates from HEK 293T cells as indicated (Lysates). Proteins were separated by SDS-PAGE and visualized by Coomassie staining or Western blot as indicated. (B) Lysates from E. coli expressing GST-SidI or GST alone were incubated with magnetic glutathione agarose beads and lysates from HEK 293T followed by 10, to 1000 µg of purified recombinant SusF (shown as increasing amounts in supernatants from beads). Proteins remaining on the beads were separated by SDS-PAGE and visualized by Coomassie stain or Western blot as indicated. GST-SidI (∼130 kDa) and SusF (∼27 kDa) are indicated with arrowheads. Data are representative of at least two independent experiments.

    Article Snippet: SDS-PAGE and Western Blot Boiled protein samples were loaded onto either 4-20% gradient SDS-PAGE gels (BioRad), 12% or 15% SDS-PAGE gels.

    Techniques: Incubation, Purification, SDS Page, Staining, Western Blot, Expressing, Recombinant

    SusF and eEF1A interact with distinct regions of SidI. (A) Schematic representation of SidI truncation proteins. (B) Lysates from E. coli expressing GST-SidI constructs were incubated with glutathione agarose beads followed by washing and incubation with lysates from HEK 293 cells stably expressing 3FLAG-SusF as indicated. Proteins were separated by SDS-PAGE and visualized by Coomassie stain (GST-SidI) or Western blot. Arrowheads indicate fusion proteins. Data are representative of three independent experiments.

    Journal: bioRxiv

    Article Title: The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

    doi: 10.1101/845313

    Figure Lengend Snippet: SusF and eEF1A interact with distinct regions of SidI. (A) Schematic representation of SidI truncation proteins. (B) Lysates from E. coli expressing GST-SidI constructs were incubated with glutathione agarose beads followed by washing and incubation with lysates from HEK 293 cells stably expressing 3FLAG-SusF as indicated. Proteins were separated by SDS-PAGE and visualized by Coomassie stain (GST-SidI) or Western blot. Arrowheads indicate fusion proteins. Data are representative of three independent experiments.

    Article Snippet: SDS-PAGE and Western Blot Boiled protein samples were loaded onto either 4-20% gradient SDS-PAGE gels (BioRad), 12% or 15% SDS-PAGE gels.

    Techniques: Expressing, Construct, Incubation, Stable Transfection, SDS Page, Staining, Western Blot

    SusF and SidI interact directly with nanomolar affinity. (A) Lysates from HEK 293 cells stably expressing 3FLAG-SusF were incubated with glutathione beads coated with either GST or GST-SidI followed by Coomassie staining for total protein and Western blotting for SusF ( α -FLAG). Arrowheads indicate GST and GST-SidI proteins. (B) Lysates from E. coli overexpressing either GST or GST-SidI were incubated with Ni-NTA beads coated with His6-SusF followed by SDS-PAGE and Coomassie staining for proteins retained on the beads. Left panel: whole cell lysates from uninduced and induced cultures of E. coli expressing GST and GST-SidI proteins; Right panel: proteins retained on Ni-NTA beads (see Materials and Methods ). (C) Chromatograms resulting from analytical scale gel-filtration separation of either SusF alone (green trace) or SusF bound to SidI (red trace). A chromatogram of known size standards is provided for reference (blue trace). (D) Binding of His6-SidI to immobilized SusF was assessed by SPR. The reference corrected sensorgram from a single-cycle experiment is shown in black, while the outcome of fitting to a two-state binding model is shown in red. The interaction is described by an apparent KD of 3.1 nM, consisting of two individual steps where kon,1 = 2.7×10 4 M -1 s -1 and koff,1 = 5.1×10-4 s -1 and kon,2 = 2.3×10 -3 s -1 and koff,2 = 4.5×10 -4 s -1 , respectively.

    Journal: bioRxiv

    Article Title: The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

    doi: 10.1101/845313

    Figure Lengend Snippet: SusF and SidI interact directly with nanomolar affinity. (A) Lysates from HEK 293 cells stably expressing 3FLAG-SusF were incubated with glutathione beads coated with either GST or GST-SidI followed by Coomassie staining for total protein and Western blotting for SusF ( α -FLAG). Arrowheads indicate GST and GST-SidI proteins. (B) Lysates from E. coli overexpressing either GST or GST-SidI were incubated with Ni-NTA beads coated with His6-SusF followed by SDS-PAGE and Coomassie staining for proteins retained on the beads. Left panel: whole cell lysates from uninduced and induced cultures of E. coli expressing GST and GST-SidI proteins; Right panel: proteins retained on Ni-NTA beads (see Materials and Methods ). (C) Chromatograms resulting from analytical scale gel-filtration separation of either SusF alone (green trace) or SusF bound to SidI (red trace). A chromatogram of known size standards is provided for reference (blue trace). (D) Binding of His6-SidI to immobilized SusF was assessed by SPR. The reference corrected sensorgram from a single-cycle experiment is shown in black, while the outcome of fitting to a two-state binding model is shown in red. The interaction is described by an apparent KD of 3.1 nM, consisting of two individual steps where kon,1 = 2.7×10 4 M -1 s -1 and koff,1 = 5.1×10-4 s -1 and kon,2 = 2.3×10 -3 s -1 and koff,2 = 4.5×10 -4 s -1 , respectively.

    Article Snippet: SDS-PAGE and Western Blot Boiled protein samples were loaded onto either 4-20% gradient SDS-PAGE gels (BioRad), 12% or 15% SDS-PAGE gels.

    Techniques: Stable Transfection, Expressing, Incubation, Staining, Western Blot, SDS Page, Filtration, Binding Assay, SPR Assay

    eEF1A interacts with the SidI-SusF complex. Lysates from E. coli expressing GST-SidI or GST alone were incubated with magnetic glutathione agarose beads and washed followed by addition of 10, 25, 50, 100, 200, 400, 800 or 1000 µg of purified recombinant SusF (shown as increasing amounts in supernatants from beads). Beads were subsequently incubated with lysates from HEK 293T cells. Proteins remaining on the beads were separated by SDS-PAGE and visualized by Coomassie stain or Western blot

    Journal: bioRxiv

    Article Title: The Legionella pneumophila metaeffector Lpg2505 (SusF) regulates SidI-mediated translation inhibition and GDP-dependent glycosyltransferase activity

    doi: 10.1101/845313

    Figure Lengend Snippet: eEF1A interacts with the SidI-SusF complex. Lysates from E. coli expressing GST-SidI or GST alone were incubated with magnetic glutathione agarose beads and washed followed by addition of 10, 25, 50, 100, 200, 400, 800 or 1000 µg of purified recombinant SusF (shown as increasing amounts in supernatants from beads). Beads were subsequently incubated with lysates from HEK 293T cells. Proteins remaining on the beads were separated by SDS-PAGE and visualized by Coomassie stain or Western blot

    Article Snippet: SDS-PAGE and Western Blot Boiled protein samples were loaded onto either 4-20% gradient SDS-PAGE gels (BioRad), 12% or 15% SDS-PAGE gels.

    Techniques: Expressing, Incubation, Purification, Recombinant, SDS Page, Staining, Western Blot

    Pup2 predominantly affects CytoQC compared to ERAD. (A) Rescue of the CytoQC-defective phenotype observed with the exogenous expression of wild-type Pup2 ( PUP2 ) in the pup2 -10 mutant. Equal numbers of each strain were spotted as described in Figure 1C with SC-Trp-His and SC-Trp-Ura plates for selection. Vector: empty vector. (B) Missense mutation in spontaneous mutant pup2 -10 is present at residue 101, replacing Leucine for Proline. (C-D) Degradation kinetics of CytoQC and ERAD substrates were determined by pulse chase analyses. Strains were pulsed with 35S-Met/Cys for 5min for Ste6 *C and Ste6 * and 10min for ∆ssPrA, CPY* and Sec61 -2, followed by chase for the indicated time points. (E) CytoQC substrate ∆ssPrA is localized predominantly in the nucleus in WT and pup2 -10 . Substrates were detected with anti-HA antibody (green). The ER and nuclear envelope were visualized with anti- Kar2 antiserum (red). Nucleus was visualized with DAPI staining. Scale bar: 2µm. (F) Accumulation of polyubiquitinated Ste6 *C and ΔssPrA was observed in pup2 -10 mutant compared to WT. Misfolded cytosolic substrates expressed in WT and pup2 -10 were immunoprecipitated (IP) by anti-HA antibody, resolved by SDS-PAGE and analyzed by immunoblotting (IB) with anti-ubiquitin antibody to detect polyubiquitinated substrates.

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Genetic Selection Based on a Ste6*C-HA-Ura3 Substrate Identifies New Cytosolic Quality Control Alleles in Saccharomyces cerevisiae

    doi: 10.1534/g3.120.401186

    Figure Lengend Snippet: Pup2 predominantly affects CytoQC compared to ERAD. (A) Rescue of the CytoQC-defective phenotype observed with the exogenous expression of wild-type Pup2 ( PUP2 ) in the pup2 -10 mutant. Equal numbers of each strain were spotted as described in Figure 1C with SC-Trp-His and SC-Trp-Ura plates for selection. Vector: empty vector. (B) Missense mutation in spontaneous mutant pup2 -10 is present at residue 101, replacing Leucine for Proline. (C-D) Degradation kinetics of CytoQC and ERAD substrates were determined by pulse chase analyses. Strains were pulsed with 35S-Met/Cys for 5min for Ste6 *C and Ste6 * and 10min for ∆ssPrA, CPY* and Sec61 -2, followed by chase for the indicated time points. (E) CytoQC substrate ∆ssPrA is localized predominantly in the nucleus in WT and pup2 -10 . Substrates were detected with anti-HA antibody (green). The ER and nuclear envelope were visualized with anti- Kar2 antiserum (red). Nucleus was visualized with DAPI staining. Scale bar: 2µm. (F) Accumulation of polyubiquitinated Ste6 *C and ΔssPrA was observed in pup2 -10 mutant compared to WT. Misfolded cytosolic substrates expressed in WT and pup2 -10 were immunoprecipitated (IP) by anti-HA antibody, resolved by SDS-PAGE and analyzed by immunoblotting (IB) with anti-ubiquitin antibody to detect polyubiquitinated substrates.

    Article Snippet: Small equal volumes of each lysate sample were resolved on a 4–15% gradient SDS-PAGE gel (Biorad) and transferred onto a nitrocellulose membrane (Biorad) for quantification and normalization of HA-tagged substrates in each sample with immunoblot.

    Techniques: Expressing, Mutagenesis, Selection, Plasmid Preparation, Pulse Chase, Staining, Immunoprecipitation, SDS Page

    CspA binds to C9 and inhibits C9 polymerization. (A) C9 binds to immobilized CspA, and the effect is dose dependent. Binding of C9 (0.01 to 20 µg/ml) to immobilized CspA (5 µg/ml) was assayed by ELISA, and bound C9 was detected with polyclonal C9 antiserum followed by HRP-conjugated anti-goat antibody. (B) Heparin affects the CspA-C9 interaction. The effect of heparin (1 to 500 µg/ml) on binding of C9 (5 µg/ml) to immobilized CspA (5 µg/ml) was assayed. (C) NaCl inhibits the CspA-C9 interaction. The effect of NaCl (0.1 to 1 M) on binding of C9 (5 µg/ml) to immobilized CspA (5 µg/ml) was assayed. The mean values from three separate experiments are shown, and error bars show SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (D) CspA inhibits polymerization of C9. ZnCl 2 induced C9 polymerization, and after incubation the samples were separated by SDS-PAGE. Following silver staining, C9 polymers and C9 monomers were identified by their mobility. C9 polymerizes in the presence of ZnCl 2 (lane 1). CspA (2.5 and 5 µg) blocks polymer formation (lanes 2 and 3). The borrelial immune evasion protein ErpC (5 µg) (lane 4) does not influence C9 polymerization. In the absence of ZnCl 2 , C9 does not form polymers (lane 5). The data shown are representative of three independent experiments.

    Journal: mBio

    Article Title: CspA from Borrelia burgdorferi Inhibits the Terminal Complement Pathway

    doi: 10.1128/mBio.00481-13

    Figure Lengend Snippet: CspA binds to C9 and inhibits C9 polymerization. (A) C9 binds to immobilized CspA, and the effect is dose dependent. Binding of C9 (0.01 to 20 µg/ml) to immobilized CspA (5 µg/ml) was assayed by ELISA, and bound C9 was detected with polyclonal C9 antiserum followed by HRP-conjugated anti-goat antibody. (B) Heparin affects the CspA-C9 interaction. The effect of heparin (1 to 500 µg/ml) on binding of C9 (5 µg/ml) to immobilized CspA (5 µg/ml) was assayed. (C) NaCl inhibits the CspA-C9 interaction. The effect of NaCl (0.1 to 1 M) on binding of C9 (5 µg/ml) to immobilized CspA (5 µg/ml) was assayed. The mean values from three separate experiments are shown, and error bars show SD. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (D) CspA inhibits polymerization of C9. ZnCl 2 induced C9 polymerization, and after incubation the samples were separated by SDS-PAGE. Following silver staining, C9 polymers and C9 monomers were identified by their mobility. C9 polymerizes in the presence of ZnCl 2 (lane 1). CspA (2.5 and 5 µg) blocks polymer formation (lanes 2 and 3). The borrelial immune evasion protein ErpC (5 µg) (lane 4) does not influence C9 polymerization. In the absence of ZnCl 2 , C9 does not form polymers (lane 5). The data shown are representative of three independent experiments.

    Article Snippet: The samples were subjected to an 8 to 16% SDS-PAGE gradient gel (Bio-Rad, Hercules, CA), and C9 polymerization was determined by silver staining.

    Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, SDS Page, Silver Staining

    Localization of the TCC inhibitory region in CspA. (A) Full-length CspA 26–251 and four deletion mutants were expressed in Escherichia coli and purified. The numbers refer to amino acid residues that are included in each construct (left). Black indicates the C7/C9 binding region, and gray indicates the factor H binding region of CspA (left). Binding of C7 (5 µg/ml) (middle) or C9 (5 µg/ml) (right) to immobilized CspA deletion mutants was assayed by ELISA. Bound C7 was detected with polyclonal C7 antiserum, and bound C9 was detected with polyclonal C9 antiserum followed by HRP-conjugated anti-goat antibody. (B and C) C7 binds to CspA with an affinity of 5.1 ± 0.2 µM and C9 with an affinity of 3.4 ± 0.1 µM. Binding of CspA, CspA 26–108 , or BSA (0.005 to 160 µM) to NT-647-labeled C7 (12.5 nM) or C9 (12.5 nM) was evaluated in fluid phase by microscale thermophoresis. Thermophoresis was recorded at 80% LED power and 80% MST power for 30 s in a Monilith NT.115 instrument. The relative fluorescence in the thermophoresis phase of the experiment has been plotted against the concentration of CspA. (D) Localization of the region within CspA that mediates TCC inhibition. Full-length CspA 26–251 and the four deletion mutants were combined with C7, C8, and C9, and thereafter the mixture was added to C5b-6-coated sheep erythrocytes. Lysis of sheep erythrocytes in the presence of C5b-9 was set to 100%. The mean values from three separate experiments are shown, and error bars show SD. ***, P ≤ 0.001. (E) The amino acid residues 109 to 251 of CspA are relevant for inhibition of C9 polymerization. ZnCl 2 induced C9 polymerization, and after incubation the samples were separated by SDS-PAGE; following silver staining, C9 polymers (pC9) and C9 monomers (mC9) were identified by their mobility. C9 polymerizes in the presence of ZnCl 2 (lane 1). CspA 26–251 , CspA 26–240 , CspA 26–215 (lanes 2 to 7), and CspA 61–251 (lanes 10 and 11) (2.5 and 5 µg) block polymer formation. CspA 26–108 (lanes 8 and 9) does not influence C9 polymerization. The data shown are representative of three independent experiments.

    Journal: mBio

    Article Title: CspA from Borrelia burgdorferi Inhibits the Terminal Complement Pathway

    doi: 10.1128/mBio.00481-13

    Figure Lengend Snippet: Localization of the TCC inhibitory region in CspA. (A) Full-length CspA 26–251 and four deletion mutants were expressed in Escherichia coli and purified. The numbers refer to amino acid residues that are included in each construct (left). Black indicates the C7/C9 binding region, and gray indicates the factor H binding region of CspA (left). Binding of C7 (5 µg/ml) (middle) or C9 (5 µg/ml) (right) to immobilized CspA deletion mutants was assayed by ELISA. Bound C7 was detected with polyclonal C7 antiserum, and bound C9 was detected with polyclonal C9 antiserum followed by HRP-conjugated anti-goat antibody. (B and C) C7 binds to CspA with an affinity of 5.1 ± 0.2 µM and C9 with an affinity of 3.4 ± 0.1 µM. Binding of CspA, CspA 26–108 , or BSA (0.005 to 160 µM) to NT-647-labeled C7 (12.5 nM) or C9 (12.5 nM) was evaluated in fluid phase by microscale thermophoresis. Thermophoresis was recorded at 80% LED power and 80% MST power for 30 s in a Monilith NT.115 instrument. The relative fluorescence in the thermophoresis phase of the experiment has been plotted against the concentration of CspA. (D) Localization of the region within CspA that mediates TCC inhibition. Full-length CspA 26–251 and the four deletion mutants were combined with C7, C8, and C9, and thereafter the mixture was added to C5b-6-coated sheep erythrocytes. Lysis of sheep erythrocytes in the presence of C5b-9 was set to 100%. The mean values from three separate experiments are shown, and error bars show SD. ***, P ≤ 0.001. (E) The amino acid residues 109 to 251 of CspA are relevant for inhibition of C9 polymerization. ZnCl 2 induced C9 polymerization, and after incubation the samples were separated by SDS-PAGE; following silver staining, C9 polymers (pC9) and C9 monomers (mC9) were identified by their mobility. C9 polymerizes in the presence of ZnCl 2 (lane 1). CspA 26–251 , CspA 26–240 , CspA 26–215 (lanes 2 to 7), and CspA 61–251 (lanes 10 and 11) (2.5 and 5 µg) block polymer formation. CspA 26–108 (lanes 8 and 9) does not influence C9 polymerization. The data shown are representative of three independent experiments.

    Article Snippet: The samples were subjected to an 8 to 16% SDS-PAGE gradient gel (Bio-Rad, Hercules, CA), and C9 polymerization was determined by silver staining.

    Techniques: Purification, Construct, Binding Assay, Enzyme-linked Immunosorbent Assay, Labeling, Microscale Thermophoresis, Fluorescence, Concentration Assay, Inhibition, Lysis, Incubation, SDS Page, Silver Staining, Blocking Assay

    DipA associates with PxdA-marked endosomes. (A) Sypro Ruby-stained SDS-PAGE gel of lysates incubated with HA-conjugated agarose from wild-type (Ctrl) or PxdA-HA expressing hyphae. Mass spectrometry identified DipA (indicated by arrow) from two biological replicates. (B) Representative kymograph of DipA-GFP movement. Also see Video 3. (C) Histogram of DipA-GFP velocities calculated from kymographs as in (B). Mean velocities are 2.44 ± 0.77 (SD) μm/sec. n = 257 moving events. (D) Colocalization of DipA-GFP and PxdA-mKate along a hypha. (E) Representative kymograph of DipA and PxdA co-movement. (F) Quantification of the percent overlap of DipA colocalized with PxdA. Mean percent overlap is 97.88 ± 6.01 (SD). N = 8 kymographs. (G) Representative image of DipA-GFP distribution in hookAΔ hyphae. (H) DipA-GFP distribution is quantified by fluorescence intensity line-scans of fluorescently-tagged organelles in wild-type (WT) or hookAΔ hyphae. Mean fluorescence intensity (solid lines) ± SEM (shading) is plotted as a function of distance from the hyphal tip. n = 3 (WT) and 7 ( hookAΔ ) hyphae.

    Journal: bioRxiv

    Article Title: Regulation of peroxisome and lipid droplet hitchhiking by PxdA and the DipA phosphatase

    doi: 10.1101/2020.02.03.932616

    Figure Lengend Snippet: DipA associates with PxdA-marked endosomes. (A) Sypro Ruby-stained SDS-PAGE gel of lysates incubated with HA-conjugated agarose from wild-type (Ctrl) or PxdA-HA expressing hyphae. Mass spectrometry identified DipA (indicated by arrow) from two biological replicates. (B) Representative kymograph of DipA-GFP movement. Also see Video 3. (C) Histogram of DipA-GFP velocities calculated from kymographs as in (B). Mean velocities are 2.44 ± 0.77 (SD) μm/sec. n = 257 moving events. (D) Colocalization of DipA-GFP and PxdA-mKate along a hypha. (E) Representative kymograph of DipA and PxdA co-movement. (F) Quantification of the percent overlap of DipA colocalized with PxdA. Mean percent overlap is 97.88 ± 6.01 (SD). N = 8 kymographs. (G) Representative image of DipA-GFP distribution in hookAΔ hyphae. (H) DipA-GFP distribution is quantified by fluorescence intensity line-scans of fluorescently-tagged organelles in wild-type (WT) or hookAΔ hyphae. Mean fluorescence intensity (solid lines) ± SEM (shading) is plotted as a function of distance from the hyphal tip. n = 3 (WT) and 7 ( hookAΔ ) hyphae.

    Article Snippet: Western blotting and mass spectrometryAll protein samples were resolved on 4-12% gradient SDS-PAGE gels (Life Technologies) for 60 mins.

    Techniques: Staining, SDS Page, Incubation, Expressing, Mass Spectrometry, Fluorescence

    Bacterially expressed MDM2-FL and MDM2-C can be recognized by distinct monoclonal antibodies. ( A ) pRSETA-mdm2-C was constructed from pRSETA-hdm2, 33 a generous gift from Dr. Lindsay Mayo, by PCR-mediated cloning. MDM2-FL and MDM2-C were overexpressed in BL21DE3 E. coli strains with plasmids pRSETA-hdm2 and pRSETA-mdm2-C, respectively. 2 µg of the bacterially purified MDM2-FL and MDM2-C, lanes 1 and 2, respectively, were loaded into 10% SDS-PAGE gel. Proteins were transferred to PVDF membrane. The quality of purification was confirmed by using either Pierce reversible staining for membrane or Coomassie Blue for in-gel detection. Arrows indicate the bands for MDM2-FL and MDM2-C. ( B ) Purified MDM2-C and ( C ) MDM2-FL proteins on a Western Blot visualized with either seconday anti-mouse or anti-rabbit antibody conjugated with Cy-3 and Cy-5. MDM2-FL-specific monoclonal antibody 2A9, or MDM2-C-specific monoclonal antibody 7C7 in combination with R D polyclonal antibody (which detects both MDM2-FL and MDM2-C). The bottom image is the superimposition of bands probed with two antibodies. ( D ) Schematic structural domains of MDM2-FL and MDM2-C. Mdm2 gene consists of 12 exons. Known motifs are represented in color, Green: p53-binding domain; Blue: Acidic domain; Purple: zinc-finger domain; Red: RING domain (really interesting new gene). The domain interface clusters and cartoon representation of the complex of MDM2-C RING and UQ_con are show (E2 Ubiquitin-Conjugating Enzyme D2) and ubiquitin are shown. This data represents results from three independent experiments.

    Journal: Cancer Management and Research

    Article Title: MDM2-C Functions as an E3 Ubiquitin Ligase

    doi: 10.2147/CMAR.S260943

    Figure Lengend Snippet: Bacterially expressed MDM2-FL and MDM2-C can be recognized by distinct monoclonal antibodies. ( A ) pRSETA-mdm2-C was constructed from pRSETA-hdm2, 33 a generous gift from Dr. Lindsay Mayo, by PCR-mediated cloning. MDM2-FL and MDM2-C were overexpressed in BL21DE3 E. coli strains with plasmids pRSETA-hdm2 and pRSETA-mdm2-C, respectively. 2 µg of the bacterially purified MDM2-FL and MDM2-C, lanes 1 and 2, respectively, were loaded into 10% SDS-PAGE gel. Proteins were transferred to PVDF membrane. The quality of purification was confirmed by using either Pierce reversible staining for membrane or Coomassie Blue for in-gel detection. Arrows indicate the bands for MDM2-FL and MDM2-C. ( B ) Purified MDM2-C and ( C ) MDM2-FL proteins on a Western Blot visualized with either seconday anti-mouse or anti-rabbit antibody conjugated with Cy-3 and Cy-5. MDM2-FL-specific monoclonal antibody 2A9, or MDM2-C-specific monoclonal antibody 7C7 in combination with R D polyclonal antibody (which detects both MDM2-FL and MDM2-C). The bottom image is the superimposition of bands probed with two antibodies. ( D ) Schematic structural domains of MDM2-FL and MDM2-C. Mdm2 gene consists of 12 exons. Known motifs are represented in color, Green: p53-binding domain; Blue: Acidic domain; Purple: zinc-finger domain; Red: RING domain (really interesting new gene). The domain interface clusters and cartoon representation of the complex of MDM2-C RING and UQ_con are show (E2 Ubiquitin-Conjugating Enzyme D2) and ubiquitin are shown. This data represents results from three independent experiments.

    Article Snippet: As high as 4–12% of gradient SDS-PAGE gel (Invitrogen) was run to separate the protein samples from in vitro ubiquitination assay, and 10% SDS-PAGE gel was run to separate the protein samples for confirming purification.

    Techniques: Construct, Polymerase Chain Reaction, Clone Assay, Purification, SDS Page, Staining, Western Blot, Binding Assay

    MDM2-C, like MDM2-FL, ubiquitinates wtp53 and mtp53 R273H.in vitro. ( A ) In vitro ubiquitination assays were performed with 2 μg of purified wild-type p53 and 1μg of recombinant MDM2-FL, MDM2-C, or untransformed negative control extract. The proteins were resolved on a 4–12% SDS-PAGE gradient gel and transferred to PVDF membrane. The primary p53 monoclonal antibody DO1 (Santa Cruz) was used to detect p53 by Western blot using Typhoon. Higher mobility bands resolved above the 53kD-band demonstrate ubiquitinated p53. ( B ) In vitro ubiquitination assays were performed with 1 μg of purified mtp53 and 1μg of recombinant MDM2-FL, MDM2-C, or untransformed negative control extract. Higher mobility bands resolved above the 53kD-band demonstrate ubiquitinated p53. This data represents results from three independent experiments.

    Journal: Cancer Management and Research

    Article Title: MDM2-C Functions as an E3 Ubiquitin Ligase

    doi: 10.2147/CMAR.S260943

    Figure Lengend Snippet: MDM2-C, like MDM2-FL, ubiquitinates wtp53 and mtp53 R273H.in vitro. ( A ) In vitro ubiquitination assays were performed with 2 μg of purified wild-type p53 and 1μg of recombinant MDM2-FL, MDM2-C, or untransformed negative control extract. The proteins were resolved on a 4–12% SDS-PAGE gradient gel and transferred to PVDF membrane. The primary p53 monoclonal antibody DO1 (Santa Cruz) was used to detect p53 by Western blot using Typhoon. Higher mobility bands resolved above the 53kD-band demonstrate ubiquitinated p53. ( B ) In vitro ubiquitination assays were performed with 1 μg of purified mtp53 and 1μg of recombinant MDM2-FL, MDM2-C, or untransformed negative control extract. Higher mobility bands resolved above the 53kD-band demonstrate ubiquitinated p53. This data represents results from three independent experiments.

    Article Snippet: As high as 4–12% of gradient SDS-PAGE gel (Invitrogen) was run to separate the protein samples from in vitro ubiquitination assay, and 10% SDS-PAGE gel was run to separate the protein samples for confirming purification.

    Techniques: In Vitro, Purification, Recombinant, Negative Control, SDS Page, Western Blot

    IFNγ-dependent engagement of mTOR effectors is Sin1-dependent. A–I , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. A–I ( top panels ), cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE and then subjected to immunoblotting with the indicated specific anti-phosphoantibodies. The blots in the respective top panels were stripped and probed with anti-AKT ( A–C ), anti-mTOR ( D and E ), anti-4E-BP1 ( F ), anti-p70S6K ( G ), anti-rpS6 ( H ), and anti-eIF4B ( I ) antibodies. J–N , serum-starved U937 cells transfected with control siRNA or Sin1 siRNA were treated with human IFNγ (5 × 10 3 IU/ml) for the indicated times. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and then processed for immunoblotting with anti- phospho-Ser-473 AKT, AKT, Sin1, and GAPDH antibodies ( J ), anti-phospho-Thr-308 AKT and anti-AKT antibodies ( K ), antibodies against phospho-Ser-2481 mTOR, mTOR, Sin1, or GAPDH ( L ), anti-phospho-Thr-37/46 4E-BP1 and anti-4E-BP1 antibodies ( M ), and anti-phospho-Thr-389 p70S6K and anti-p70S6K antibodies ( N ). A–K ( bottom panels ), bands from three independent experiments (including the blots shown) were quantified by densitometry. Data are expressed as ratios of phosphoprotein over respective total protein values, and bar graphs represent means ± S.E. ( error bars ) of three independent experiments for each experimental condition.

    Journal: The Journal of Biological Chemistry

    Article Title: Central Regulatory Role for SIN1 in Interferon γ (IFNγ) Signaling and Generation of Biological Responses *

    doi: 10.1074/jbc.M116.757666

    Figure Lengend Snippet: IFNγ-dependent engagement of mTOR effectors is Sin1-dependent. A–I , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. A–I ( top panels ), cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE and then subjected to immunoblotting with the indicated specific anti-phosphoantibodies. The blots in the respective top panels were stripped and probed with anti-AKT ( A–C ), anti-mTOR ( D and E ), anti-4E-BP1 ( F ), anti-p70S6K ( G ), anti-rpS6 ( H ), and anti-eIF4B ( I ) antibodies. J–N , serum-starved U937 cells transfected with control siRNA or Sin1 siRNA were treated with human IFNγ (5 × 10 3 IU/ml) for the indicated times. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and then processed for immunoblotting with anti- phospho-Ser-473 AKT, AKT, Sin1, and GAPDH antibodies ( J ), anti-phospho-Thr-308 AKT and anti-AKT antibodies ( K ), antibodies against phospho-Ser-2481 mTOR, mTOR, Sin1, or GAPDH ( L ), anti-phospho-Thr-37/46 4E-BP1 and anti-4E-BP1 antibodies ( M ), and anti-phospho-Thr-389 p70S6K and anti-p70S6K antibodies ( N ). A–K ( bottom panels ), bands from three independent experiments (including the blots shown) were quantified by densitometry. Data are expressed as ratios of phosphoprotein over respective total protein values, and bar graphs represent means ± S.E. ( error bars ) of three independent experiments for each experimental condition.

    Article Snippet: For immunoblotting analyses, lysates were resolved by SDS-PAGE gradient gels (Bio-Rad), transferred to Immobilon-P PVDF membranes (Millipore), which were probed with primary and secondary antibodies, and then detected by enhanced chemiluminescence as in previous studies ( , ).

    Techniques: SDS Page, Transfection

    IFNγ-induced tyrosine phosphorylation of STAT1 is Sin1-dependent. A and B , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. Cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE and then subjected to immunoblotting analyses with the indicated antibodies. A and B ( bottom panels ), bands from three independent experiments (including the blots shown) were quantified by densitometry. Data are expressed as ratios of phospho-Stat1 over total Stat1, and bar graphs represent means ± S.E. ( error bars ) of three independent experiments for each experimental condition. C–E , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. C , equal amounts of protein were processed for immunoprecipitation ( IP ) with anti-IFNGR1 antibody or control IgG, as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-IFNGR1, anti-Sin1, and anti-Stat1 antibodies. D and E , equal amounts of protein were processed for immunoprecipitation with anti-Jak1 ( D ) or anti-Jak2 ( E ) and control IgG, as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-phospho-Tyr-1022/1023 Jak1, Jak1, and Sin1 antibodies (D) or with anti-phospho-Tyr-1007/1008 Jak2, Jak2, and Sin1 antibodies ( E ). F , serum-starved mLST8 +/− and mLST8 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. Equal amounts of protein were processed for immunoprecipitation with anti-IFNGR1 antibody or control IgG as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-IFNGR1, Sin1, Jak1, and Stat1 antibodies. G , the same cell lysates used in H were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-phospho-Tyr-701 Stat1, Stat1, mLST8, Sin1, and GAPDH antibodies.

    Journal: The Journal of Biological Chemistry

    Article Title: Central Regulatory Role for SIN1 in Interferon γ (IFNγ) Signaling and Generation of Biological Responses *

    doi: 10.1074/jbc.M116.757666

    Figure Lengend Snippet: IFNγ-induced tyrosine phosphorylation of STAT1 is Sin1-dependent. A and B , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. Cell lysates were prepared, and equal amounts of protein were resolved by SDS-PAGE and then subjected to immunoblotting analyses with the indicated antibodies. A and B ( bottom panels ), bands from three independent experiments (including the blots shown) were quantified by densitometry. Data are expressed as ratios of phospho-Stat1 over total Stat1, and bar graphs represent means ± S.E. ( error bars ) of three independent experiments for each experimental condition. C–E , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. C , equal amounts of protein were processed for immunoprecipitation ( IP ) with anti-IFNGR1 antibody or control IgG, as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-IFNGR1, anti-Sin1, and anti-Stat1 antibodies. D and E , equal amounts of protein were processed for immunoprecipitation with anti-Jak1 ( D ) or anti-Jak2 ( E ) and control IgG, as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-phospho-Tyr-1022/1023 Jak1, Jak1, and Sin1 antibodies (D) or with anti-phospho-Tyr-1007/1008 Jak2, Jak2, and Sin1 antibodies ( E ). F , serum-starved mLST8 +/− and mLST8 −/− MEFs were treated with mouse IFNγ (5 × 10 3 IU/ml) for the indicated times. Equal amounts of protein were processed for immunoprecipitation with anti-IFNGR1 antibody or control IgG as indicated. The immunoprecipitated proteins were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-IFNGR1, Sin1, Jak1, and Stat1 antibodies. G , the same cell lysates used in H were resolved by SDS-PAGE and then subjected to immunoblot analyses with anti-phospho-Tyr-701 Stat1, Stat1, mLST8, Sin1, and GAPDH antibodies.

    Article Snippet: For immunoblotting analyses, lysates were resolved by SDS-PAGE gradient gels (Bio-Rad), transferred to Immobilon-P PVDF membranes (Millipore), which were probed with primary and secondary antibodies, and then detected by enhanced chemiluminescence as in previous studies ( , ).

    Techniques: SDS Page, Immunoprecipitation

    Requirement of Sin1 for type II IFN-induced ISG protein expression. A and B , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (1.5 × 10 3 IU/ml) as indicated. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and immunoblotted with anti-IP10 ( A ) or anti-DAPK1 ( B ). C and D , serum-starved U937 cells transfected with control siRNA or Sin1 siRNA were treated with human IFNγ (1.5 × 10 3 IU/ml) for the indicated times. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and then processed for immunoblotting with anti-IP10 ( C ) and anti-DAPK1 and anti-SLFN5 ( D ) antibodies. A–D , anti-GAPDH antibody was used for loading control.

    Journal: The Journal of Biological Chemistry

    Article Title: Central Regulatory Role for SIN1 in Interferon γ (IFNγ) Signaling and Generation of Biological Responses *

    doi: 10.1074/jbc.M116.757666

    Figure Lengend Snippet: Requirement of Sin1 for type II IFN-induced ISG protein expression. A and B , serum-starved Sin1 +/+ and Sin1 −/− MEFs were treated with mouse IFNγ (1.5 × 10 3 IU/ml) as indicated. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and immunoblotted with anti-IP10 ( A ) or anti-DAPK1 ( B ). C and D , serum-starved U937 cells transfected with control siRNA or Sin1 siRNA were treated with human IFNγ (1.5 × 10 3 IU/ml) for the indicated times. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and then processed for immunoblotting with anti-IP10 ( C ) and anti-DAPK1 and anti-SLFN5 ( D ) antibodies. A–D , anti-GAPDH antibody was used for loading control.

    Article Snippet: For immunoblotting analyses, lysates were resolved by SDS-PAGE gradient gels (Bio-Rad), transferred to Immobilon-P PVDF membranes (Millipore), which were probed with primary and secondary antibodies, and then detected by enhanced chemiluminescence as in previous studies ( , ).

    Techniques: Expressing, SDS Page, Transfection

    Restricting CDKL5 expression to the cell nucleus. ( A ) Extracts of CDKL5 disrupted U–2–OS (Flp-In T-Rex) cells ( CDKL5 Δ/Δ ) stably expressing CDKL5 NLS –WT or a K 42 R kinase-dead mutant (CDKL5 NLS –KD) or empty vector were subjected to western blotting with the antibodies indicated. Two different dishes of cells are shown per condition. ( B ) CDKL5 Δ/Δ cells stably expressing CDKL5, CDKL5 NLS –WT or CDKL5 NLS –KD were subjected to indirect immunofluorescence analysis with anti-CDKL5 antibodies. ( C ) Subcellular fractionation of lysates from CDKL5 ΄/Δ cells stably expressing CDKL5, CDKL5 NLS –WT or CDKL5 NLS –KD or empty vector. Lysates were fractionated to isolate proteins found in the following subcellular compartments: cytoplasmic (Cyt), membrane (Mb), nuclear (Nuc), chromatin (Chr) or cytoskeleton (Csk). Fractionated samples were resolved by SDS-PAGE and probed with antibodies shown. ( D ) CDKL5 Δ/Δ cells stably expressing CDKL5 NLS –WT or CDKL5 NLS –KD (or empty vector) were treated with 500 μM H 2 O 2 for 15 min. Samples were resolved by SDS-PAGE and probed with indicated antibodies or stained with Ponceau S to show equal loading. Rep=biological replicate.

    Journal: bioRxiv

    Article Title: Epilepsy kinase CDKL5 is a DNA damage sensor which controls transcriptional activity at DNA breaks

    doi: 10.1101/2020.12.10.419747

    Figure Lengend Snippet: Restricting CDKL5 expression to the cell nucleus. ( A ) Extracts of CDKL5 disrupted U–2–OS (Flp-In T-Rex) cells ( CDKL5 Δ/Δ ) stably expressing CDKL5 NLS –WT or a K 42 R kinase-dead mutant (CDKL5 NLS –KD) or empty vector were subjected to western blotting with the antibodies indicated. Two different dishes of cells are shown per condition. ( B ) CDKL5 Δ/Δ cells stably expressing CDKL5, CDKL5 NLS –WT or CDKL5 NLS –KD were subjected to indirect immunofluorescence analysis with anti-CDKL5 antibodies. ( C ) Subcellular fractionation of lysates from CDKL5 ΄/Δ cells stably expressing CDKL5, CDKL5 NLS –WT or CDKL5 NLS –KD or empty vector. Lysates were fractionated to isolate proteins found in the following subcellular compartments: cytoplasmic (Cyt), membrane (Mb), nuclear (Nuc), chromatin (Chr) or cytoskeleton (Csk). Fractionated samples were resolved by SDS-PAGE and probed with antibodies shown. ( D ) CDKL5 Δ/Δ cells stably expressing CDKL5 NLS –WT or CDKL5 NLS –KD (or empty vector) were treated with 500 μM H 2 O 2 for 15 min. Samples were resolved by SDS-PAGE and probed with indicated antibodies or stained with Ponceau S to show equal loading. Rep=biological replicate.

    Article Snippet: Samples were resolved in 4–12% Bis–Tris SDS–PAGE gradient gels (Nupage, Thermo Fisher) and relevant bands were excised and further processed for mass spectrometry as detailed below.

    Techniques: Expressing, Stable Transfection, Mutagenesis, Plasmid Preparation, Western Blot, Immunofluorescence, Fractionation, SDS Page, Staining

    Validating phosphorylation of EP400, ELOA and TTDN1. ( A ) HEK293 cells were co-transfected with CDKL5 NLS (wild type “WT” or kinase-dead “KD” K 42 R mutant) and either FLAG-EP400 (left), FLAG-ELOA (middle) or FLAG-TTDN1 (right). 24 hr later cells were incubated with H 2 O 2 (500 μM) for 15 min before being harvested and lysed. Protein extracts were subjected to immunoprecipitation with anti-FLAG-agarose beads. Precipitates were subjected to SDS-PAGE and blotting with antibodies shown (bottom panels) or staining with Coomassie Brilliant Blue (top panels). The bands corresponding to the FLAG-tagged proteins were excised from the gels in A. and processed for mass spectrometric detection of relevant phospho-peptides. Three independent co-transfection experiments were done for every condition. ( B ) Boxplots showing VSN–normalised intensity of phospho-peptides corresponding to EP400 pSer 729 , ELOA pSer 311 and TTDN1 pSer 40 from the experiment in A. ( C ) Boxplots of the VSN-adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG–EP400, FLAG–ELOA and FLAG–TTDN1 from the experiment in A. ( D ) Left : Anti-FLAG precipitates from HEK293 cells transiently expressing FLAG-tagged CDKL5 (wild type “WT” or a K 42 R kinase-dead “KD” mutant) were incubated with the synthetic peptides indicated, in the presence of [γ- 32 P]-labelled ATP-Mg 2+ and peptide phosphorylation was measured by Cerenkov counting. Data are represented as mean ± SEM from three independent experiments. Right : Same but anti-FLAG precipitates were subjected to SDS-PAGE and autoradiography to detect CDKL5 autophosphorylation, or western blotting with CDKL5-pTyr 171 antibody specific for the CDKL5-Tyr171 autophosphorylation site ( 2 ).

    Journal: bioRxiv

    Article Title: Epilepsy kinase CDKL5 is a DNA damage sensor which controls transcriptional activity at DNA breaks

    doi: 10.1101/2020.12.10.419747

    Figure Lengend Snippet: Validating phosphorylation of EP400, ELOA and TTDN1. ( A ) HEK293 cells were co-transfected with CDKL5 NLS (wild type “WT” or kinase-dead “KD” K 42 R mutant) and either FLAG-EP400 (left), FLAG-ELOA (middle) or FLAG-TTDN1 (right). 24 hr later cells were incubated with H 2 O 2 (500 μM) for 15 min before being harvested and lysed. Protein extracts were subjected to immunoprecipitation with anti-FLAG-agarose beads. Precipitates were subjected to SDS-PAGE and blotting with antibodies shown (bottom panels) or staining with Coomassie Brilliant Blue (top panels). The bands corresponding to the FLAG-tagged proteins were excised from the gels in A. and processed for mass spectrometric detection of relevant phospho-peptides. Three independent co-transfection experiments were done for every condition. ( B ) Boxplots showing VSN–normalised intensity of phospho-peptides corresponding to EP400 pSer 729 , ELOA pSer 311 and TTDN1 pSer 40 from the experiment in A. ( C ) Boxplots of the VSN-adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG–EP400, FLAG–ELOA and FLAG–TTDN1 from the experiment in A. ( D ) Left : Anti-FLAG precipitates from HEK293 cells transiently expressing FLAG-tagged CDKL5 (wild type “WT” or a K 42 R kinase-dead “KD” mutant) were incubated with the synthetic peptides indicated, in the presence of [γ- 32 P]-labelled ATP-Mg 2+ and peptide phosphorylation was measured by Cerenkov counting. Data are represented as mean ± SEM from three independent experiments. Right : Same but anti-FLAG precipitates were subjected to SDS-PAGE and autoradiography to detect CDKL5 autophosphorylation, or western blotting with CDKL5-pTyr 171 antibody specific for the CDKL5-Tyr171 autophosphorylation site ( 2 ).

    Article Snippet: Samples were resolved in 4–12% Bis–Tris SDS–PAGE gradient gels (Nupage, Thermo Fisher) and relevant bands were excised and further processed for mass spectrometry as detailed below.

    Techniques: Transfection, Mutagenesis, Incubation, Immunoprecipitation, SDS Page, Staining, Cotransfection, Expressing, Autoradiography, Western Blot

    Dynamin GTPases are modestly phosphorylated by LRRK2 in vitro. In vitro kinase assays with [ 33 P]-γ-ATP, soluble recombinant full-length FLAG-tagged LRRK2 variants (WT, G2019S or D1994A) and immunopurified ‘on-bead’ YFP-tagged ArfGAP1 ( A ), GFP-tagged Dnm1 ( B ), and Myc-tagged Drp1 ( C ), Mfn1 ( D ), Mfn2 ( E ) or OPA1 ( F ), derived by IP from transfected HEK-293T cells. Following kinase reactions, soluble LRRK2 and ‘on-bead’ substrates were separated and resolved on independent SDS–PAGE gels, as indicated. Western blot analysis with anti-GFP, anti-myc or anti-FLAG antibodies indicate equal loading of ArfGAP1, Dnm1, Drp1, Mfn1, Mfn2, OPA1 and LRRK2 proteins in each condition. Autoradiographs ( 33 P) reveal the LRRK2-dependent phosphorylation of ArfGAP1, Dnm1, Drp1, Mfn1 and OPA1, with enhanced phosphorylation by G2019S LRRK2 compared with WT or kinase-inactive D1994A LRRK2. A soluble eluate from FLAG IPs (derived from non-transfected cells) was used as a control in each assay to assess background 33 P incorporation for each substrate. LRRK2 autophosphorylation is also detected in these assays. Blots are representative of at least three-independent kinase experiments.

    Journal: Human Molecular Genetics

    Article Title: Functional interaction of Parkinson's disease-associated LRRK2 with members of the dynamin GTPase superfamily

    doi: 10.1093/hmg/ddt600

    Figure Lengend Snippet: Dynamin GTPases are modestly phosphorylated by LRRK2 in vitro. In vitro kinase assays with [ 33 P]-γ-ATP, soluble recombinant full-length FLAG-tagged LRRK2 variants (WT, G2019S or D1994A) and immunopurified ‘on-bead’ YFP-tagged ArfGAP1 ( A ), GFP-tagged Dnm1 ( B ), and Myc-tagged Drp1 ( C ), Mfn1 ( D ), Mfn2 ( E ) or OPA1 ( F ), derived by IP from transfected HEK-293T cells. Following kinase reactions, soluble LRRK2 and ‘on-bead’ substrates were separated and resolved on independent SDS–PAGE gels, as indicated. Western blot analysis with anti-GFP, anti-myc or anti-FLAG antibodies indicate equal loading of ArfGAP1, Dnm1, Drp1, Mfn1, Mfn2, OPA1 and LRRK2 proteins in each condition. Autoradiographs ( 33 P) reveal the LRRK2-dependent phosphorylation of ArfGAP1, Dnm1, Drp1, Mfn1 and OPA1, with enhanced phosphorylation by G2019S LRRK2 compared with WT or kinase-inactive D1994A LRRK2. A soluble eluate from FLAG IPs (derived from non-transfected cells) was used as a control in each assay to assess background 33 P incorporation for each substrate. LRRK2 autophosphorylation is also detected in these assays. Blots are representative of at least three-independent kinase experiments.

    Article Snippet: Reactions samples were resolved on 3–8% Tris–acetate or 4–16% Tris–glycine SDS–PAGE gradient gels (Invitrogen) and transferred to PVDF membranes.

    Techniques: In Vitro, Recombinant, Derivative Assay, Transfection, SDS Page, Western Blot

    Subcellular distribution and native complexes of dynamin GTPases are not altered by LRRK2 in mouse brain. ( A ) Subcellular fractionation of cerebral cortex tissue derived from human G2019S LRRK2 transgenic and non-transgenic mice. Dnm1 and Drp1 are broadly distributed across multiple membrane and soluble fractions, whereas Mfn2 and OPA1 are enriched in heavy membrane (P2) and synaptosomal membrane (LP1) fractions. LRRK2 is broadly detected with enrichment in light membrane/microsomal (P3), synaptosomal LP1 and synaptic vesicle-enriched (LP2) membrane fractions. The distribution of the synaptic vesicle-associated protein, synaptophysin 1, demonstrates enrichment of membranes in P2, P3, LP1 and LP2 fractions, whereas Mfn2 and OPA1 indicate enrichment of mitochondria in P2 and LP1 fractions. ( B ) Native-PAGE and ( C ) SDS–PAGE analysis of equivalent cerebral cortex extracts derived from human G2019S LRRK2 transgenic (Tg) and non-transgenic (NTg) mice, and LRRK2 KO and WT mice, revealing similar oligomeric protein complexes for Dnm1, Drp1 and OPA1. (C) LRRK2 antibodies confirm the absence of LRRK2 in KO mice (mouse-selective N241A/34 antibody) and human G2019S LRRK2 expression in transgenic mice (human-selective MJFF4/c81-8 antibody; lower band = LRRK2; asterisk indicates non-specific upper band). ( D and E ) Size-exclusion chromatography on soluble whole brain extracts from WT and LRRK2 KO mice. Sequential fractions (#1–16, 0.5 ml) and total homogenates (WT or KO) were analyzed by western blotting with antibodies to Dnm1, Drp1 and LRRK2 (N241A/34), or β-tubulin as a loading control. The elution profile of Dnm1 and Drp1 is similar in WT and KO brains, whereas the elution profile of individual standards is indicated. LRRK2 antibody (N241A/34) confirms the absence of LRRK2 in KO mice. Blots are representative of duplicate experiments. Molecular mass markers are indicated in kDa.

    Journal: Human Molecular Genetics

    Article Title: Functional interaction of Parkinson's disease-associated LRRK2 with members of the dynamin GTPase superfamily

    doi: 10.1093/hmg/ddt600

    Figure Lengend Snippet: Subcellular distribution and native complexes of dynamin GTPases are not altered by LRRK2 in mouse brain. ( A ) Subcellular fractionation of cerebral cortex tissue derived from human G2019S LRRK2 transgenic and non-transgenic mice. Dnm1 and Drp1 are broadly distributed across multiple membrane and soluble fractions, whereas Mfn2 and OPA1 are enriched in heavy membrane (P2) and synaptosomal membrane (LP1) fractions. LRRK2 is broadly detected with enrichment in light membrane/microsomal (P3), synaptosomal LP1 and synaptic vesicle-enriched (LP2) membrane fractions. The distribution of the synaptic vesicle-associated protein, synaptophysin 1, demonstrates enrichment of membranes in P2, P3, LP1 and LP2 fractions, whereas Mfn2 and OPA1 indicate enrichment of mitochondria in P2 and LP1 fractions. ( B ) Native-PAGE and ( C ) SDS–PAGE analysis of equivalent cerebral cortex extracts derived from human G2019S LRRK2 transgenic (Tg) and non-transgenic (NTg) mice, and LRRK2 KO and WT mice, revealing similar oligomeric protein complexes for Dnm1, Drp1 and OPA1. (C) LRRK2 antibodies confirm the absence of LRRK2 in KO mice (mouse-selective N241A/34 antibody) and human G2019S LRRK2 expression in transgenic mice (human-selective MJFF4/c81-8 antibody; lower band = LRRK2; asterisk indicates non-specific upper band). ( D and E ) Size-exclusion chromatography on soluble whole brain extracts from WT and LRRK2 KO mice. Sequential fractions (#1–16, 0.5 ml) and total homogenates (WT or KO) were analyzed by western blotting with antibodies to Dnm1, Drp1 and LRRK2 (N241A/34), or β-tubulin as a loading control. The elution profile of Dnm1 and Drp1 is similar in WT and KO brains, whereas the elution profile of individual standards is indicated. LRRK2 antibody (N241A/34) confirms the absence of LRRK2 in KO mice. Blots are representative of duplicate experiments. Molecular mass markers are indicated in kDa.

    Article Snippet: Reactions samples were resolved on 3–8% Tris–acetate or 4–16% Tris–glycine SDS–PAGE gradient gels (Invitrogen) and transferred to PVDF membranes.

    Techniques: Fractionation, Derivative Assay, Transgenic Assay, Mouse Assay, Clear Native PAGE, SDS Page, Expressing, Size-exclusion Chromatography, Western Blot

    SDS–PAGE and a ligand blot analysis of the HaABCC1 fragments. (A) Marker, protein marker; Lane 1, TMD1 fragment after ultrasound treatment; Lane 2, TMD2 fragment after ultrasound treatment. (B) Lanes 1 and 4, protein marker; Lanes 3 and 6, bovine serum albumin (BSA), as a control; Lane 2, TMD1 fragment bound with Cry2Ab; Lane 5 TMD2 fragment bound with Cry2Ab.

    Journal: Frontiers in Physiology

    Article Title: Specific Binding Protein ABCC1 Is Associated With Cry2Ab Toxicity in Helicoverpa armigera

    doi: 10.3389/fphys.2018.00745

    Figure Lengend Snippet: SDS–PAGE and a ligand blot analysis of the HaABCC1 fragments. (A) Marker, protein marker; Lane 1, TMD1 fragment after ultrasound treatment; Lane 2, TMD2 fragment after ultrasound treatment. (B) Lanes 1 and 4, protein marker; Lanes 3 and 6, bovine serum albumin (BSA), as a control; Lane 2, TMD1 fragment bound with Cry2Ab; Lane 5 TMD2 fragment bound with Cry2Ab.

    Article Snippet: Ligand Blot Analyses For the ligand blot analysis, 10 μg purified HaABCC1 fragments were separated using 4–20% gradient SDS-PAGE gels (Genscript Biology Co., NJ, United States) and then electro-transferred onto PVDF filters (Millipore Corp.).

    Techniques: SDS Page, Marker

    Sindbis virus (SINV) virion purification strategy and production flowchart. (A) Clarified viral preparations were purified over two potassium tartrate density gradients done in series using isopycnic ultracentrifugation and a final wash and pellet step. Negative controls (uninfected cell culture supernatant) were processed in parallel with the virus preparations. (B) Transmission electron micrographs were taken of the purified virions to confirm efficiency of this enrichment procedure. (C) An SDS-PAGE gel was stained with silver or with Coomassie blue for each of the viral preparations. HepG2 preparations are presented here and are representative of all viral preparations from all host cellular backgrounds.

    Journal: Journal of Virology

    Article Title: Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virion and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication

    doi: 10.1128/JVI.00694-18

    Figure Lengend Snippet: Sindbis virus (SINV) virion purification strategy and production flowchart. (A) Clarified viral preparations were purified over two potassium tartrate density gradients done in series using isopycnic ultracentrifugation and a final wash and pellet step. Negative controls (uninfected cell culture supernatant) were processed in parallel with the virus preparations. (B) Transmission electron micrographs were taken of the purified virions to confirm efficiency of this enrichment procedure. (C) An SDS-PAGE gel was stained with silver or with Coomassie blue for each of the viral preparations. HepG2 preparations are presented here and are representative of all viral preparations from all host cellular backgrounds.

    Article Snippet: Additionally, to check for copurified protein contaminants, each preparation was resolved on a 4 to 12% Bis-Tris SDS-PAGE gradient gel (Invitrogen) as described previously ( ) and stained with silver in the method of Wray et al. ( ) or with Coomassie blue.

    Techniques: Purification, Cell Culture, Transmission Assay, SDS Page, Staining

    Subcellular fractionation of mouse cerebral cortex . (A) Schematic representation of the subcellular fractionation steps. P1, nuclear pellet and debris; P2, crude synaptosomal fraction; P3, light membranes; S3, cytosolic fraction; LP1, synaptosomal membrane fraction; LP2, synaptic vesicle-enriched fraction; LS2, soluble synaptosomal fraction. (B) Western blots of JNPL3 and non-tg male mouse cerebral cortex subcellular fractions. P1, P2, P3, or S3 fraction derived from 0.13 mg wet weight of tissue and LP1, LP2, or LS2 fraction derived from 0.5 mg wet weight of tissue were loaded on SDS-PAGE. Blots were probed with PSD-95, synaptotagmin, GAP-43, Tau5, E1, β-tubulin, and β-actin antibodies. (C) Proportions of protein levels in fractions (P1, P2, P3, and S3) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice are shown. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (P1 + P2 + P3 + S3). Results are expressed as mean ± SEM. (D) Proportions of protein levels in synaptosomal fractions (LP1, LP2, and LS2) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice were indicated. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (LP1 + LP2 + LS2). Results are expressed as mean ± SEM.

    Journal: Frontiers in Neurology

    Article Title: Biochemical Distribution of Tau Protein in Synaptosomal Fraction of Transgenic Mice Expressing Human P301L Tau

    doi: 10.3389/fneur.2014.00026

    Figure Lengend Snippet: Subcellular fractionation of mouse cerebral cortex . (A) Schematic representation of the subcellular fractionation steps. P1, nuclear pellet and debris; P2, crude synaptosomal fraction; P3, light membranes; S3, cytosolic fraction; LP1, synaptosomal membrane fraction; LP2, synaptic vesicle-enriched fraction; LS2, soluble synaptosomal fraction. (B) Western blots of JNPL3 and non-tg male mouse cerebral cortex subcellular fractions. P1, P2, P3, or S3 fraction derived from 0.13 mg wet weight of tissue and LP1, LP2, or LS2 fraction derived from 0.5 mg wet weight of tissue were loaded on SDS-PAGE. Blots were probed with PSD-95, synaptotagmin, GAP-43, Tau5, E1, β-tubulin, and β-actin antibodies. (C) Proportions of protein levels in fractions (P1, P2, P3, and S3) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice are shown. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (P1 + P2 + P3 + S3). Results are expressed as mean ± SEM. (D) Proportions of protein levels in synaptosomal fractions (LP1, LP2, and LS2) of tau (Tau5) and β-tubulin (β-tub) from JNPL3 ( n = 5) and non-tg ( n = 5) mice were indicated. Intensities of tau (49–65 kDa) and β-tubulin (50 kDa) were measured by Bio-Imaging Analyzer System. Ratios were indicated by percent of total (LP1 + LP2 + LS2). Results are expressed as mean ± SEM.

    Article Snippet: The samples were separated by gel electrophoresis on 10 or 5–20% gradient SDS-PAGE gels (Wako Pure Chemical Industries, Osaka, Japan), and transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany).

    Techniques: Fractionation, Western Blot, Derivative Assay, SDS Page, Mouse Assay, Imaging

    Soluble and sarkosyl-insoluble tau in JNPL3 male mice . (A) Western blots of TBS-soluble tau in mouse cerebral cortices. Equal volumes of TBS-soluble fraction derived from 0.2 mg wet weight of brain from eight male JNPL3 and two male non-tg mice were separated by SDS-PAGE, blotted, and then probed with E1, Tau5, MS06, and GAPDH antibodies. (B) Western blot of sarkosyl-insoluble fractions in mouse cerebral cortices. Samples derived from 20 mg wet weight from male JNPL3 and non-tg mice, 5 mg wet weight from female JNPL3 cortex (ctx), and 2.5 mg wet weight from female JNPL3 spinal cord (SPc) were separated by SDS-PAGE, blotted, and then probed with E1 antibody.

    Journal: Frontiers in Neurology

    Article Title: Biochemical Distribution of Tau Protein in Synaptosomal Fraction of Transgenic Mice Expressing Human P301L Tau

    doi: 10.3389/fneur.2014.00026

    Figure Lengend Snippet: Soluble and sarkosyl-insoluble tau in JNPL3 male mice . (A) Western blots of TBS-soluble tau in mouse cerebral cortices. Equal volumes of TBS-soluble fraction derived from 0.2 mg wet weight of brain from eight male JNPL3 and two male non-tg mice were separated by SDS-PAGE, blotted, and then probed with E1, Tau5, MS06, and GAPDH antibodies. (B) Western blot of sarkosyl-insoluble fractions in mouse cerebral cortices. Samples derived from 20 mg wet weight from male JNPL3 and non-tg mice, 5 mg wet weight from female JNPL3 cortex (ctx), and 2.5 mg wet weight from female JNPL3 spinal cord (SPc) were separated by SDS-PAGE, blotted, and then probed with E1 antibody.

    Article Snippet: The samples were separated by gel electrophoresis on 10 or 5–20% gradient SDS-PAGE gels (Wako Pure Chemical Industries, Osaka, Japan), and transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany).

    Techniques: Mouse Assay, Western Blot, Derivative Assay, SDS Page

    Quantitative western blot analysis of tau protein . (A) Tau band patterns in cytosolic (S3) and synaptosomal membrane (LP1) fractions from four JNPL3 and four non-tg mouse cerebral cortices. Equal volumes of fractions derived from 0.25 mg wet weight of brain were separated by SDS-PAGE, blotted, and then probed with Tau5, MS06, Tau1, pS199, pT231, and pS396 antibodies. (B,C) The relative ratio of tau protein between S3 and LP1 fractions from JNPL3 (B) and non-tg (C) mice was measured ( n = 4 each). Results are expressed as mean ± SEM. The mean value of tau protein in S3 fraction from JNPL3 mice was normalized to one.

    Journal: Frontiers in Neurology

    Article Title: Biochemical Distribution of Tau Protein in Synaptosomal Fraction of Transgenic Mice Expressing Human P301L Tau

    doi: 10.3389/fneur.2014.00026

    Figure Lengend Snippet: Quantitative western blot analysis of tau protein . (A) Tau band patterns in cytosolic (S3) and synaptosomal membrane (LP1) fractions from four JNPL3 and four non-tg mouse cerebral cortices. Equal volumes of fractions derived from 0.25 mg wet weight of brain were separated by SDS-PAGE, blotted, and then probed with Tau5, MS06, Tau1, pS199, pT231, and pS396 antibodies. (B,C) The relative ratio of tau protein between S3 and LP1 fractions from JNPL3 (B) and non-tg (C) mice was measured ( n = 4 each). Results are expressed as mean ± SEM. The mean value of tau protein in S3 fraction from JNPL3 mice was normalized to one.

    Article Snippet: The samples were separated by gel electrophoresis on 10 or 5–20% gradient SDS-PAGE gels (Wako Pure Chemical Industries, Osaka, Japan), and transferred to nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany).

    Techniques: Western Blot, Derivative Assay, SDS Page, Mouse Assay

    GelE cleavage of rAce analyzed by SDS-PAGE. Sample lanes from left to right represent reaction mixtures containing 30 μg of rAce/ml with various amounts of purified GelE (0.015, 0.045, 0.15, 0.45. 1.5, 4.5, and 15 μg/ml), rAce (30 μg/ml)

    Journal: Journal of Bacteriology

    Article Title: The Fsr Quorum-Sensing System of Enterococcus faecalisModulates Surface Display of the Collagen-Binding MSCRAMM Ace through Regulation of gelE ▿

    doi: 10.1128/JB.05026-11

    Figure Lengend Snippet: GelE cleavage of rAce analyzed by SDS-PAGE. Sample lanes from left to right represent reaction mixtures containing 30 μg of rAce/ml with various amounts of purified GelE (0.015, 0.045, 0.15, 0.45. 1.5, 4.5, and 15 μg/ml), rAce (30 μg/ml)

    Article Snippet: Samples were applied onto SDS-PAGE gradient gels (4 to 20%) (NuSep, Inc.), followed with Coomassie blue protein staining (Sigma).

    Techniques: SDS Page, Purification