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    Structured Review

    Millipore streptavidin agarose beads
    PRMT5 peptide pull-downs confirm an in vitro interaction with NHERF2. A , GST-fused PDZ domains of GRIP1 (residues 672–754), MPP7 (residues 139–220), PDZ-LIM55 (residues 2–85), NHERF1 FL (residues 1–355), NHERF2 FL (residues 1–337), SCRIB (residues 714–801), PDZ-LIM2 (residues 1–84), and GST were incubated with biotinylated PRMT5 C terminus unphosphorylated peptide. Bound proteins were detected with α-GST antibody (short and long exposure are shown). Peptide loading was assessed with HRP-conjugated <t>streptavidin</t> ( SA-HRP ). The Coomassie stain demonstrates roughly equal input of the GST fusion proteins. B , schematic representation of the constructs used for peptide pull-down in C. C , purified recombinant GST, GST-tagged human NHERF2 full-length (NHERF2-PDZ 1–2), PDZ1 (amino acids 1–152), PDZ2 (amino acids 107–337), and 14-3-3ϵ were incubated with biotinylated PRMT5 C terminus unphosphorylated and Thr-634-phosphorylated peptides bound to streptavidin-agarose beads and detected by α-GST. Left lane , inputs of the GST fusion proteins. D , 293T cells were transfected with constructs expressing GFP-14-3-3ϵ and Myc-PRMT5 wild type or T634A mutant. Cell lysates were then incubated with normal mouse IgG or α-Myc antibody. Immunocomplexes were captured by Protein A beads and detected by either α-Myc or α-GFP. IB , immunoblotting.
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    Images

    1) Product Images from "PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch *"

    Article Title: PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.760330

    PRMT5 peptide pull-downs confirm an in vitro interaction with NHERF2. A , GST-fused PDZ domains of GRIP1 (residues 672–754), MPP7 (residues 139–220), PDZ-LIM55 (residues 2–85), NHERF1 FL (residues 1–355), NHERF2 FL (residues 1–337), SCRIB (residues 714–801), PDZ-LIM2 (residues 1–84), and GST were incubated with biotinylated PRMT5 C terminus unphosphorylated peptide. Bound proteins were detected with α-GST antibody (short and long exposure are shown). Peptide loading was assessed with HRP-conjugated streptavidin ( SA-HRP ). The Coomassie stain demonstrates roughly equal input of the GST fusion proteins. B , schematic representation of the constructs used for peptide pull-down in C. C , purified recombinant GST, GST-tagged human NHERF2 full-length (NHERF2-PDZ 1–2), PDZ1 (amino acids 1–152), PDZ2 (amino acids 107–337), and 14-3-3ϵ were incubated with biotinylated PRMT5 C terminus unphosphorylated and Thr-634-phosphorylated peptides bound to streptavidin-agarose beads and detected by α-GST. Left lane , inputs of the GST fusion proteins. D , 293T cells were transfected with constructs expressing GFP-14-3-3ϵ and Myc-PRMT5 wild type or T634A mutant. Cell lysates were then incubated with normal mouse IgG or α-Myc antibody. Immunocomplexes were captured by Protein A beads and detected by either α-Myc or α-GFP. IB , immunoblotting.
    Figure Legend Snippet: PRMT5 peptide pull-downs confirm an in vitro interaction with NHERF2. A , GST-fused PDZ domains of GRIP1 (residues 672–754), MPP7 (residues 139–220), PDZ-LIM55 (residues 2–85), NHERF1 FL (residues 1–355), NHERF2 FL (residues 1–337), SCRIB (residues 714–801), PDZ-LIM2 (residues 1–84), and GST were incubated with biotinylated PRMT5 C terminus unphosphorylated peptide. Bound proteins were detected with α-GST antibody (short and long exposure are shown). Peptide loading was assessed with HRP-conjugated streptavidin ( SA-HRP ). The Coomassie stain demonstrates roughly equal input of the GST fusion proteins. B , schematic representation of the constructs used for peptide pull-down in C. C , purified recombinant GST, GST-tagged human NHERF2 full-length (NHERF2-PDZ 1–2), PDZ1 (amino acids 1–152), PDZ2 (amino acids 107–337), and 14-3-3ϵ were incubated with biotinylated PRMT5 C terminus unphosphorylated and Thr-634-phosphorylated peptides bound to streptavidin-agarose beads and detected by α-GST. Left lane , inputs of the GST fusion proteins. D , 293T cells were transfected with constructs expressing GFP-14-3-3ϵ and Myc-PRMT5 wild type or T634A mutant. Cell lysates were then incubated with normal mouse IgG or α-Myc antibody. Immunocomplexes were captured by Protein A beads and detected by either α-Myc or α-GFP. IB , immunoblotting.

    Techniques Used: In Vitro, Incubation, Staining, Construct, Purification, Recombinant, Transfection, Expressing, Mutagenesis

    2) Product Images from "Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress"

    Article Title: Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

    Journal: Journal of Virology

    doi: 10.1128/JVI.00812-17

    Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with streptavidin beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).
    Figure Legend Snippet: Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with streptavidin beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).

    Techniques Used: Mutagenesis, Western Blot, Expressing

    3) Product Images from "Oxidized Phospholipid Inhibition of Toll-like Receptor (TLR) Signaling Is Restricted to TLR2 and TLR4"

    Article Title: Oxidized Phospholipid Inhibition of Toll-like Receptor (TLR) Signaling Is Restricted to TLR2 and TLR4

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M800352200

    Effect of OxPAPC on binding of LPS to MD2. A , supernatant of cells transfected with MD2-FLAG was exposed to biotinylated LPS ( B-LPS ) with or without 15 min preincubation with unlabeled LPS or 50 μg/ml OxPAPC. LPS-MD2 complexes were precipitated with streptavidin-agarose beads, resuspended in SDS-PAGE loading buffer, and separated on 15% SDS-PAGE gels before transfer to nitrocellulose blots. Precipitated MD2 was visualized with anti-FLAG antibody. B , mean intensity of precipitated MD2 bands was determined by densitometry from three experiments ± S.D., p
    Figure Legend Snippet: Effect of OxPAPC on binding of LPS to MD2. A , supernatant of cells transfected with MD2-FLAG was exposed to biotinylated LPS ( B-LPS ) with or without 15 min preincubation with unlabeled LPS or 50 μg/ml OxPAPC. LPS-MD2 complexes were precipitated with streptavidin-agarose beads, resuspended in SDS-PAGE loading buffer, and separated on 15% SDS-PAGE gels before transfer to nitrocellulose blots. Precipitated MD2 was visualized with anti-FLAG antibody. B , mean intensity of precipitated MD2 bands was determined by densitometry from three experiments ± S.D., p

    Techniques Used: Binding Assay, Transfection, SDS Page

    4) Product Images from "Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10"

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    Journal: Glycobiology

    doi: 10.1093/glycob/cwr097

    The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.
    Figure Legend Snippet: The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Techniques Used: Expressing, Synthesized, Labeling, Radioactivity, Incubation, Immunoprecipitation, Electrophoresis, Autoradiography

    Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.
    Figure Legend Snippet: Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Techniques Used: Inhibition, Expressing, Labeling, Western Blot

    5) Product Images from "Analysis of 2?-phosphotransferase (Tpt1p) from Saccharomyces cerevisiae: Evidence for a conserved two-step reaction mechanism"

    Article Title: Analysis of 2?-phosphotransferase (Tpt1p) from Saccharomyces cerevisiae: Evidence for a conserved two-step reaction mechanism

    Journal: RNA

    doi: 10.1261/rna.7194605

    Intermediate formed with biotin-NAD can be resolved on streptavidin beads. Reaction mixtures containing 1 nM p*ApA p pA substrate, 12 μM Tpt1 K69A/R71S protein, and 75 μM NAD (lanes b – d ) or biotin NAD (lanes e – l )
    Figure Legend Snippet: Intermediate formed with biotin-NAD can be resolved on streptavidin beads. Reaction mixtures containing 1 nM p*ApA p pA substrate, 12 μM Tpt1 K69A/R71S protein, and 75 μM NAD (lanes b – d ) or biotin NAD (lanes e – l )

    Techniques Used: TNKS1 Histone Ribosylation Assay

    6) Product Images from "Regulation of SNAREs by tomosyn and ROCK"

    Article Title: Regulation of SNAREs by tomosyn and ROCK

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200405002

    Inhibition of vesicle transport by tomosyn and ROCK. (A) Transport of VSV-G in NG108 cells. NG108 cells were cotransfected with GFP-VSV-G and either HA-tomosyn, myc-ROCK-Δ3 (ROCK-CA), or a null HA plasmid (Control). At 4 h after the transfection, the cells were incubated at 20°C for 2 h. Parallel samples were transferred to 37°C immediately after the 20°C incubation. The cotransfected cells were identified by the expression of GFP (green) and either immunostaining of HA or myc (red), and the distributions of GFP-VSV-G were examined. Bars, 20 μm. (B) Kinetics of VSV-G cell surface transport in NG108 cells. NG108 cells were cotransfected with VSV-G and either HA-tomosyn, myc-ROCK-Δ3 (ROCK-CA), or a null HA plasmid (Control). At 4 h after the transfection, the cells were labeled with [ 35 S]methionine and were incubated for indicated periods of time. To detect cell surface VSV-G, the cell surface was biotinylated. After the biotinylation, the cells were lysed and total VSV-G was collected by immunoprecipitation with the anti-VSV-G mAb. Cell surface–biotinylated VSV-G was recovered with streptavidin-agarose beads from total VSV-G. Biotinylated (S: surface) and total (T: 20% of total) VSV-G were separated by SDS-PAGE and the intensity of each band was quantified. (C) Quantification of the formation of SNARE and tomosyn complexes in NG108 cells. NG108 cells were transfected with HA-tomosyn, myc-ROCK-Δ3 (ROCK-CA), or a null HA plasmid (Control), cultured in DME containing 1 mM db-cAMP for 48 h, and allowed to extend neurites. Cells were lysed and immunoprecipitated with the anti-syntaxin-1 mAb or the control mouse IgG, followed by immunoblotting with the indicated antibodies. The quantification of immunoblot is shown on the bottom.
    Figure Legend Snippet: Inhibition of vesicle transport by tomosyn and ROCK. (A) Transport of VSV-G in NG108 cells. NG108 cells were cotransfected with GFP-VSV-G and either HA-tomosyn, myc-ROCK-Δ3 (ROCK-CA), or a null HA plasmid (Control). At 4 h after the transfection, the cells were incubated at 20°C for 2 h. Parallel samples were transferred to 37°C immediately after the 20°C incubation. The cotransfected cells were identified by the expression of GFP (green) and either immunostaining of HA or myc (red), and the distributions of GFP-VSV-G were examined. Bars, 20 μm. (B) Kinetics of VSV-G cell surface transport in NG108 cells. NG108 cells were cotransfected with VSV-G and either HA-tomosyn, myc-ROCK-Δ3 (ROCK-CA), or a null HA plasmid (Control). At 4 h after the transfection, the cells were labeled with [ 35 S]methionine and were incubated for indicated periods of time. To detect cell surface VSV-G, the cell surface was biotinylated. After the biotinylation, the cells were lysed and total VSV-G was collected by immunoprecipitation with the anti-VSV-G mAb. Cell surface–biotinylated VSV-G was recovered with streptavidin-agarose beads from total VSV-G. Biotinylated (S: surface) and total (T: 20% of total) VSV-G were separated by SDS-PAGE and the intensity of each band was quantified. (C) Quantification of the formation of SNARE and tomosyn complexes in NG108 cells. NG108 cells were transfected with HA-tomosyn, myc-ROCK-Δ3 (ROCK-CA), or a null HA plasmid (Control), cultured in DME containing 1 mM db-cAMP for 48 h, and allowed to extend neurites. Cells were lysed and immunoprecipitated with the anti-syntaxin-1 mAb or the control mouse IgG, followed by immunoblotting with the indicated antibodies. The quantification of immunoblot is shown on the bottom.

    Techniques Used: Inhibition, Plasmid Preparation, Transfection, Incubation, Expressing, Immunostaining, Labeling, Immunoprecipitation, SDS Page, Cell Culture

    7) Product Images from "LncRNA DANCR upregulates PI3K/AKT signaling through activating serine phosphorylation of RXRA"

    Article Title: LncRNA DANCR upregulates PI3K/AKT signaling through activating serine phosphorylation of RXRA

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-1220-7

    DANCR interacts with RXRA in TNBC cells. a Schematic diagram of putative RXRA binding sites in DANCR . b RIP-qPCR assay of the association of RXRA with DANCR in MDA-MB-231 and MDA-MB-468 cells. c Re-expression of shRNA-resistant DANCR wild type and RXRA-binding mutant types. d RIP-qPCR assay of effects of re-expression of shRNA-resistant DANCR wild type or mutant types on RXRA binding. e Biotinylated DANCR was incubated with nuclear extracts (MDA-MB-231 and MDA-MB-468 cells), targeted with streptavidin beads, and binding proteins were resolved in a gel. Western blotting assay of the specific binding of RXRA and DANCR . f , g RNAs corresponding to fragments in different regions of DANCR were treated as in ( e ), and binding RXRA was detected by western blotting assay. Error bars ± SD. * P
    Figure Legend Snippet: DANCR interacts with RXRA in TNBC cells. a Schematic diagram of putative RXRA binding sites in DANCR . b RIP-qPCR assay of the association of RXRA with DANCR in MDA-MB-231 and MDA-MB-468 cells. c Re-expression of shRNA-resistant DANCR wild type and RXRA-binding mutant types. d RIP-qPCR assay of effects of re-expression of shRNA-resistant DANCR wild type or mutant types on RXRA binding. e Biotinylated DANCR was incubated with nuclear extracts (MDA-MB-231 and MDA-MB-468 cells), targeted with streptavidin beads, and binding proteins were resolved in a gel. Western blotting assay of the specific binding of RXRA and DANCR . f , g RNAs corresponding to fragments in different regions of DANCR were treated as in ( e ), and binding RXRA was detected by western blotting assay. Error bars ± SD. * P

    Techniques Used: Binding Assay, Real-time Polymerase Chain Reaction, Multiple Displacement Amplification, Expressing, shRNA, Mutagenesis, Incubation, Western Blot

    8) Product Images from "Structure of the parathyroid hormone receptor C-terminus bound to the G-protein dimer G?1?2"

    Article Title: Structure of the parathyroid hormone receptor C-terminus bound to the G-protein dimer G?1?2

    Journal:

    doi: 10.1016/j.str.2008.04.010

    Direct binding of Gβ 1 γ 2 to the PTH1R C-terminal tail peptide. Purified Gβ 1 γ 2 protein at indicated concentrations was injected over a streptavidin SPR biosensor surface, previously coated with biotin-PTH1R peptide (aa 466-487),
    Figure Legend Snippet: Direct binding of Gβ 1 γ 2 to the PTH1R C-terminal tail peptide. Purified Gβ 1 γ 2 protein at indicated concentrations was injected over a streptavidin SPR biosensor surface, previously coated with biotin-PTH1R peptide (aa 466-487),

    Techniques Used: Binding Assay, Purification, Injection, SPR Assay

    9) Product Images from "Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions"

    Article Title: Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

    Journal: Journal of Virology

    doi: 10.1128/JVI.01258-13

    A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins
    Figure Legend Snippet: A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins

    Techniques Used: Western Blot, Infection

    Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The
    Figure Legend Snippet: Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The

    Techniques Used: Expressing, Construct, Recombinant, Binding Assay

    10) Product Images from "H3K27me1 is essential for MMP-9-dependent H3N-terminal tail proteolysis during osteoclastogenesis"

    Article Title: H3K27me1 is essential for MMP-9-dependent H3N-terminal tail proteolysis during osteoclastogenesis

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0193-1

    MMP-9 binding to H3K27me1 nucleosomes. a Schematic depiction of the domain structure of MMP-9. b Peptide pull-down assays with biotinylated H3 1–21 and 21–44 peptides and recombinant His-MMP-9 were analyzed by Western blotting with anti-His antibody. H3 peptides were unmodified, K18ac or K27me1 as indicated. Lane 1 represents 10% of the input MMP-9. c Nucleosomes were reconstituted on a 207-bp 601 nucleosome positioning sequence using unmodified or H3K27me1 histone octamers and immobilized on streptavidin beads. His-MMP-9 was incubated with immobilized nucleosomes, and its binding to nucleosomes was analyzed by Western blotting with anti-His antibody. Lane 1 contains 10% of the input MMP-9. d H3K27me1 nucleosomes were incubated with immobilized MMP-9 N-terminal (amino acids 112–447) and C-terminal (amino acids 448–730) domains. After extensive washing, the binding of H3K27me1 nucleosomes to MMP-9 domains was determined by Western blotting with anti-H3 antibody. Input corresponds to 10% of H3K27me1 nucleosomes used in the binding reactions. e After incubation with H3K27me1 nucleosomes, the binding of MMP-9N-terminal subregions to nucleosomes was determined by Western blotting with anti-His antibody. Input lanes 1–3 represent 10% of MMP-9 fragments used in the binding reactions. f OCP-induced cells were transfected with FLAG-H3 wild type (WT) or K27R mutant (K27R), and mononucleosomes were prepared by micrococcal nuclease digestion as summarized in Figure S3. Mononucleosomes containing ectopic H3 were immunoprecipitated from total mononucleosomes with FLAG antibody and analyzed by Western blotting with anti-MMP-9 antibody. g Model of the MMP-9-H3K27me1 interaction. PDB entries 4h3x (mMMP-9) and 3avr (H3.1) were used in docking simulations using the program Cluspro 2.0 [ 28 – 30 ]. Simulations were run with non-methylated H3. For context, H3K27 is shown monomethylated. h Nucleosome binding assays were conducted as in e , except that His-MMP-9 amino acids 384–447 carrying E402A mutation were used
    Figure Legend Snippet: MMP-9 binding to H3K27me1 nucleosomes. a Schematic depiction of the domain structure of MMP-9. b Peptide pull-down assays with biotinylated H3 1–21 and 21–44 peptides and recombinant His-MMP-9 were analyzed by Western blotting with anti-His antibody. H3 peptides were unmodified, K18ac or K27me1 as indicated. Lane 1 represents 10% of the input MMP-9. c Nucleosomes were reconstituted on a 207-bp 601 nucleosome positioning sequence using unmodified or H3K27me1 histone octamers and immobilized on streptavidin beads. His-MMP-9 was incubated with immobilized nucleosomes, and its binding to nucleosomes was analyzed by Western blotting with anti-His antibody. Lane 1 contains 10% of the input MMP-9. d H3K27me1 nucleosomes were incubated with immobilized MMP-9 N-terminal (amino acids 112–447) and C-terminal (amino acids 448–730) domains. After extensive washing, the binding of H3K27me1 nucleosomes to MMP-9 domains was determined by Western blotting with anti-H3 antibody. Input corresponds to 10% of H3K27me1 nucleosomes used in the binding reactions. e After incubation with H3K27me1 nucleosomes, the binding of MMP-9N-terminal subregions to nucleosomes was determined by Western blotting with anti-His antibody. Input lanes 1–3 represent 10% of MMP-9 fragments used in the binding reactions. f OCP-induced cells were transfected with FLAG-H3 wild type (WT) or K27R mutant (K27R), and mononucleosomes were prepared by micrococcal nuclease digestion as summarized in Figure S3. Mononucleosomes containing ectopic H3 were immunoprecipitated from total mononucleosomes with FLAG antibody and analyzed by Western blotting with anti-MMP-9 antibody. g Model of the MMP-9-H3K27me1 interaction. PDB entries 4h3x (mMMP-9) and 3avr (H3.1) were used in docking simulations using the program Cluspro 2.0 [ 28 – 30 ]. Simulations were run with non-methylated H3. For context, H3K27 is shown monomethylated. h Nucleosome binding assays were conducted as in e , except that His-MMP-9 amino acids 384–447 carrying E402A mutation were used

    Techniques Used: Binding Assay, Recombinant, Western Blot, Sequencing, Incubation, Transfection, Mutagenesis, Immunoprecipitation, Methylation

    11) Product Images from "Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease"

    Article Title: Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI71651

    Coimmunostaining of DPPIV and LAMP2a in CaCo2-BBE cells with redistribution of DPPIV to large vesicles in MYO5B-KD cells. ( A – L ) x-y confocal images are shown above z-axis reconstructions. Control and MYO5B-KD cells were immunostained for DPPIV (green) and LAMP2a (red). ( A – F ) Control cells showed a normal apical distribution of DPPIV and diffusely cytoplasmic LAMP2a. ( G – L ) In MYO5B-KD cells, DPPIV was seen in internal vesicles, some of which also stained for LAMP2a (white arrow). ( M ) Magnified x-z image from control cells in F . ( N ) Magnified x-z image from MYO5B-KD cells in L showing cytoplasmic distribution of DPPIV. ( O ) Control cells immunostained for DPPIV (green) and RAB11A (red) showed apical DPPIV and subapical RAB11A. ( P – R ) MYO5B-KD cells, immunostained for DPPIV (green) and RAB11A (red), showed dispersal of RAB11A from the subapical surface and localization in large DPPIV positive vesicles (white arrows). ( S ) DPPIV reduction in MYO5B-KD cells shown via mean fluorescence in maximum-intensity Z-stack projections. ( T ) Western blot comparing control and MYO5B-KD cell lines probed for DPPIV and α-tubulin demonstrating reduction of DPPIV expression. ( U ) Apical surface biotinylation in control and MYO5B-KD cells showing DPPIV immunoreactivity in total protein, flow through from streptavidin beads, and biotinylated streptavidin-bound protein, demonstrating an increase in the nonbiotinylated cytoplasmic pool and a decrease in DPPIV on the apical surface in the MYO5B-KD (Bound-MVBKD) versus control (Bound-Ctrl) cells. Scale bar: 10 μm. * P ≤ 0.05, Mann-Whitney test. Error bars denote mean ± SEM.
    Figure Legend Snippet: Coimmunostaining of DPPIV and LAMP2a in CaCo2-BBE cells with redistribution of DPPIV to large vesicles in MYO5B-KD cells. ( A – L ) x-y confocal images are shown above z-axis reconstructions. Control and MYO5B-KD cells were immunostained for DPPIV (green) and LAMP2a (red). ( A – F ) Control cells showed a normal apical distribution of DPPIV and diffusely cytoplasmic LAMP2a. ( G – L ) In MYO5B-KD cells, DPPIV was seen in internal vesicles, some of which also stained for LAMP2a (white arrow). ( M ) Magnified x-z image from control cells in F . ( N ) Magnified x-z image from MYO5B-KD cells in L showing cytoplasmic distribution of DPPIV. ( O ) Control cells immunostained for DPPIV (green) and RAB11A (red) showed apical DPPIV and subapical RAB11A. ( P – R ) MYO5B-KD cells, immunostained for DPPIV (green) and RAB11A (red), showed dispersal of RAB11A from the subapical surface and localization in large DPPIV positive vesicles (white arrows). ( S ) DPPIV reduction in MYO5B-KD cells shown via mean fluorescence in maximum-intensity Z-stack projections. ( T ) Western blot comparing control and MYO5B-KD cell lines probed for DPPIV and α-tubulin demonstrating reduction of DPPIV expression. ( U ) Apical surface biotinylation in control and MYO5B-KD cells showing DPPIV immunoreactivity in total protein, flow through from streptavidin beads, and biotinylated streptavidin-bound protein, demonstrating an increase in the nonbiotinylated cytoplasmic pool and a decrease in DPPIV on the apical surface in the MYO5B-KD (Bound-MVBKD) versus control (Bound-Ctrl) cells. Scale bar: 10 μm. * P ≤ 0.05, Mann-Whitney test. Error bars denote mean ± SEM.

    Techniques Used: Staining, Fluorescence, Western Blot, Expressing, Flow Cytometry, MANN-WHITNEY

    Microvillus inclusions in CaCo2-BBE cells arise from internalization of the apical surface. x-y confocal images of MYO5B-YE/QR–expressing MYO5B-KD cells are shown above z-axis reconstructions. ( A – C ) CaCo2-BBE cells were stained with phalloidin (green). Images in A and B use corresponding x-y and different z-axis reconstructions from the same field of view. The z-axis reconstruction in A shows the development of an apical invagination, whereas that in B shows a nearly completed microvillus inclusion. ( C ) A separate field of view in which the z-axis reconstruction demonstrates a completely internalized microvillus inclusion. ( D – F ) The apical surface of CaCo2-BBE cells was biotinylated and fixed after 24 hours. These cells were then stained with phalloidin (green) and fluorescent streptavidin (red). Single asterisks indicate the position for the first z-axis reconstruction directly below the x-y confocal image. Double asterisks indicate the position for the second z-axis reconstruction directly below the first z-axis reconstruction. Fluorescent streptavidin was observed in microvillus inclusions inside the cells. ( G – I ) CaCo2-BBE cells from A and B double labeled with both phalloidin and SNX18 showed SNX18 localization at the bottom of a forming microvillus inclusion (white arrows), while more mature microvillus inclusions showed dispersal of SNX18 (red arrows). Scale bar: 10 μm.
    Figure Legend Snippet: Microvillus inclusions in CaCo2-BBE cells arise from internalization of the apical surface. x-y confocal images of MYO5B-YE/QR–expressing MYO5B-KD cells are shown above z-axis reconstructions. ( A – C ) CaCo2-BBE cells were stained with phalloidin (green). Images in A and B use corresponding x-y and different z-axis reconstructions from the same field of view. The z-axis reconstruction in A shows the development of an apical invagination, whereas that in B shows a nearly completed microvillus inclusion. ( C ) A separate field of view in which the z-axis reconstruction demonstrates a completely internalized microvillus inclusion. ( D – F ) The apical surface of CaCo2-BBE cells was biotinylated and fixed after 24 hours. These cells were then stained with phalloidin (green) and fluorescent streptavidin (red). Single asterisks indicate the position for the first z-axis reconstruction directly below the x-y confocal image. Double asterisks indicate the position for the second z-axis reconstruction directly below the first z-axis reconstruction. Fluorescent streptavidin was observed in microvillus inclusions inside the cells. ( G – I ) CaCo2-BBE cells from A and B double labeled with both phalloidin and SNX18 showed SNX18 localization at the bottom of a forming microvillus inclusion (white arrows), while more mature microvillus inclusions showed dispersal of SNX18 (red arrows). Scale bar: 10 μm.

    Techniques Used: Expressing, Staining, Labeling

    Loss of MYO5B in CaCo2-BBE cells causes the redistribution of basolateral markers. ( A – H ) x-y confocal images are shown above z-axis reconstructions. ( A ) p120 staining in controls showed a lateral distribution, ( B ) while MYO5B-KD cells showed decreased p120 at the lateral membranes. ( C ) Na/K-ATPase staining in controls showed a lateral distribution, ( D ) while MYO5B-KD cells showed reduction in lateral membranes. ( E ) E-cadherin staining in controls showed a junctional distribution, ( I ) with the x-z image magnified in junctional E-cadherin (white arrows). ( F ) MYO5B-KD showed redistribution of E-cadherin along the apical and lateral membranes and with internal pools of E-cadherin distributed throughout the cells; x-z image magnified in J . ( G and H ) In control and MYO5B-KD cells, β-catenin stained lateral membranes. ( K ) Quantitation of basolateral mean fluorescence. MYO5B-KD cells showed a reduction of p120 and an increase in E-cadherin. ( L and M ) Western blot with quantitation of basolateral markers in MYO5B-KD showed an isoform switch of p120, with no decrease and an increase E-cadherin total protein. ( N ) Quantitation of apical-to-basolateral ratio in MYO5B-KD cells showed a redistribution of Na/K-ATPase to the apical surface, while E-cadherin was localized over both the apical and basolateral surfaces. ( O ) Surface biotinylation of either the apical or basolateral surfaces in control and MYO5B-KD cells with total protein, flow through from streptavidin beads (FT), and biotinylated-streptavidin-bound protein from control (Bound-Ctrl) or MYO5B-KD (Bound-MVBKD) cells showed an increase in apical and a decrease in basolateral E-cadherin in the MYO5B-KD cells. Scale bar: 10 μm. * P ≤ 0.05, ** P ≤ 0.01, Mann-Whitney test. Error bars denote mean ± SEM.
    Figure Legend Snippet: Loss of MYO5B in CaCo2-BBE cells causes the redistribution of basolateral markers. ( A – H ) x-y confocal images are shown above z-axis reconstructions. ( A ) p120 staining in controls showed a lateral distribution, ( B ) while MYO5B-KD cells showed decreased p120 at the lateral membranes. ( C ) Na/K-ATPase staining in controls showed a lateral distribution, ( D ) while MYO5B-KD cells showed reduction in lateral membranes. ( E ) E-cadherin staining in controls showed a junctional distribution, ( I ) with the x-z image magnified in junctional E-cadherin (white arrows). ( F ) MYO5B-KD showed redistribution of E-cadherin along the apical and lateral membranes and with internal pools of E-cadherin distributed throughout the cells; x-z image magnified in J . ( G and H ) In control and MYO5B-KD cells, β-catenin stained lateral membranes. ( K ) Quantitation of basolateral mean fluorescence. MYO5B-KD cells showed a reduction of p120 and an increase in E-cadherin. ( L and M ) Western blot with quantitation of basolateral markers in MYO5B-KD showed an isoform switch of p120, with no decrease and an increase E-cadherin total protein. ( N ) Quantitation of apical-to-basolateral ratio in MYO5B-KD cells showed a redistribution of Na/K-ATPase to the apical surface, while E-cadherin was localized over both the apical and basolateral surfaces. ( O ) Surface biotinylation of either the apical or basolateral surfaces in control and MYO5B-KD cells with total protein, flow through from streptavidin beads (FT), and biotinylated-streptavidin-bound protein from control (Bound-Ctrl) or MYO5B-KD (Bound-MVBKD) cells showed an increase in apical and a decrease in basolateral E-cadherin in the MYO5B-KD cells. Scale bar: 10 μm. * P ≤ 0.05, ** P ≤ 0.01, Mann-Whitney test. Error bars denote mean ± SEM.

    Techniques Used: Staining, Quantitation Assay, Fluorescence, Western Blot, Flow Cytometry, MANN-WHITNEY

    12) Product Images from "Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1"

    Article Title: Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg817

    CDC5L peptides associate with PLRG1 in HeLa nuclear extract. CDC5L peptides were used in pull-down assays on streptavidin–agarose beads and the co-precipitated protein transferred to the nitrocellulose membranes was probed with anti-PLRG1 antibodies. Protein bands were then revealed by enhanced chemiluminescence (ECL). ( A ) Lane 1, a positive control containing HeLa nuclear extract; lanes 2 and 3, control pull-downs using beads only and CD-R24 peptide, respectively; lanes 4–6, protein from pull-down assays using the CD24-1, CD24-2 and CD24-3 peptides, respectively. ( B ) Pull-down assays using 12mer CDC5L peptides. Lane 1, a control containing HeLa nuclear extract; lane 2, a control pull-down assay using the CD-R12 peptide; lanes 3–5, protein from pull-down assays using the CD12-1, CD12-2 and CD12-3 peptides, respectively. ( C ) Binding of CDC5L peptides to PLRG1 in nuclear extract does not disrupt the CDC5L–PLRG1 interaction. Pull-down assays were performed as above using streptavidin–agarose beads except that the blots were probed with a buffer containing both anti-PLRG1 and anti-CDC5L antibodies. Lane 1, the positive control (HeLa nuclear extract); lanes 2 and 3, control pull-downs using the CD-R12 and CD-R24 peptides, respectively; lanes 4 and 5, pull-downs performed using the CD12-3 and CD24-1 peptides, respectively. The arrowheads on the right of the figure point to the bands representing PLRG1 or CDC5L on the nitrocellulose membrane.
    Figure Legend Snippet: CDC5L peptides associate with PLRG1 in HeLa nuclear extract. CDC5L peptides were used in pull-down assays on streptavidin–agarose beads and the co-precipitated protein transferred to the nitrocellulose membranes was probed with anti-PLRG1 antibodies. Protein bands were then revealed by enhanced chemiluminescence (ECL). ( A ) Lane 1, a positive control containing HeLa nuclear extract; lanes 2 and 3, control pull-downs using beads only and CD-R24 peptide, respectively; lanes 4–6, protein from pull-down assays using the CD24-1, CD24-2 and CD24-3 peptides, respectively. ( B ) Pull-down assays using 12mer CDC5L peptides. Lane 1, a control containing HeLa nuclear extract; lane 2, a control pull-down assay using the CD-R12 peptide; lanes 3–5, protein from pull-down assays using the CD12-1, CD12-2 and CD12-3 peptides, respectively. ( C ) Binding of CDC5L peptides to PLRG1 in nuclear extract does not disrupt the CDC5L–PLRG1 interaction. Pull-down assays were performed as above using streptavidin–agarose beads except that the blots were probed with a buffer containing both anti-PLRG1 and anti-CDC5L antibodies. Lane 1, the positive control (HeLa nuclear extract); lanes 2 and 3, control pull-downs using the CD-R12 and CD-R24 peptides, respectively; lanes 4 and 5, pull-downs performed using the CD12-3 and CD24-1 peptides, respectively. The arrowheads on the right of the figure point to the bands representing PLRG1 or CDC5L on the nitrocellulose membrane.

    Techniques Used: Positive Control, Pull Down Assay, Binding Assay

    PLRG1 peptides will interact with CDC5L in nuclear extract and inhibit pre-mRNA splicing. ( A ) Design of peptides from sequences in the CDC5L binding region of PLRG1. The arrows indicate the sequences of the peptides synthesised. ( B ) Autoradiograph of a splicing gel from an experiment to determine the effect of 24mer–30mer peptides spanning the highly conserved WD40 sequences on splicing. Approximately 7–20 nmol peptide were added to the splicing reactions (lanes 4–12). Lane 1 contained the input pre-mRNA. CTRL1 is a control splicing reaction without peptide. CTRL2 is a control reaction containing 20 nmol control peptide HC-2 derived from another spliceosomal protein HCF-1 that has not been detected in complexes containing CDC5L and PLRG1. The symbols on the right of the panel represent the input RNA, splicing intermediates and products. ( C ) Autoradiograph of a splicing gel from an experiment to determine the effect of overlapping 15mer peptides spanning the PL30-3 sequence on splicing. Similar amounts of peptide were added (lanes 4–12) to the splicing reactions as in (B). The lanes marked CTRL1 and CTRL2 contained splicing reactions treated in a similar way to lanes with the same names in (B). ( D ) Peptides containing the same amino acids as PL15-3 and PL30-3 in a scrambled sequence do not inhibit splicing. Lane 1, CTRL1 is the control reaction without peptide; lane 2, ∼20 nmol PL30-3; lanes 3–5, 7–20 nmol PL-SB15; lanes 6–8, 7–20 nmol PL-SB30 peptide. ( E ) Pull-down of CDC5L onto streptavidin–agarose beads from HeLa nuclear extract using PLRG1 peptides. Lanes 4–9, pull-down assays with the corresponding peptides (used in marking each lane). CTRL1 did not contain any peptides whereas CTRL2 contained a control peptide that does not inhibit splicing. The blot was probed with a buffer containing both anti-CDC5L and anti-PLRG1 antibodies. The arrows on the right of the panel show the positions of the CDC5L and PLRG1 proteins.
    Figure Legend Snippet: PLRG1 peptides will interact with CDC5L in nuclear extract and inhibit pre-mRNA splicing. ( A ) Design of peptides from sequences in the CDC5L binding region of PLRG1. The arrows indicate the sequences of the peptides synthesised. ( B ) Autoradiograph of a splicing gel from an experiment to determine the effect of 24mer–30mer peptides spanning the highly conserved WD40 sequences on splicing. Approximately 7–20 nmol peptide were added to the splicing reactions (lanes 4–12). Lane 1 contained the input pre-mRNA. CTRL1 is a control splicing reaction without peptide. CTRL2 is a control reaction containing 20 nmol control peptide HC-2 derived from another spliceosomal protein HCF-1 that has not been detected in complexes containing CDC5L and PLRG1. The symbols on the right of the panel represent the input RNA, splicing intermediates and products. ( C ) Autoradiograph of a splicing gel from an experiment to determine the effect of overlapping 15mer peptides spanning the PL30-3 sequence on splicing. Similar amounts of peptide were added (lanes 4–12) to the splicing reactions as in (B). The lanes marked CTRL1 and CTRL2 contained splicing reactions treated in a similar way to lanes with the same names in (B). ( D ) Peptides containing the same amino acids as PL15-3 and PL30-3 in a scrambled sequence do not inhibit splicing. Lane 1, CTRL1 is the control reaction without peptide; lane 2, ∼20 nmol PL30-3; lanes 3–5, 7–20 nmol PL-SB15; lanes 6–8, 7–20 nmol PL-SB30 peptide. ( E ) Pull-down of CDC5L onto streptavidin–agarose beads from HeLa nuclear extract using PLRG1 peptides. Lanes 4–9, pull-down assays with the corresponding peptides (used in marking each lane). CTRL1 did not contain any peptides whereas CTRL2 contained a control peptide that does not inhibit splicing. The blot was probed with a buffer containing both anti-CDC5L and anti-PLRG1 antibodies. The arrows on the right of the panel show the positions of the CDC5L and PLRG1 proteins.

    Techniques Used: Binding Assay, Autoradiography, Derivative Assay, Sequencing

    13) Product Images from "Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function *"

    Article Title: Tyrosines in the Carboxyl Terminus Regulate Syk Kinase Activity and Function *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.134262

    Binding of Syk mutants to phosphorylated γITAM. Cell lysates were prepared in the presence or absence of vanadate from non-stimulated cells 48 h after transfection with the indicated constructs. After incubation for 4 h at 4 °C to allow dephosphorylation of Syk, vanadate was added to all the samples. Lysates were then precipitated with non-phosphorylated (γ YY ) or phosphorylated (γ PP ) ITAM-biotinylated peptides pre-bound to streptavidin-agarose beads. The precipitates were analyzed by immunoblotting with anti-Syk. A , analysis of Syk-3F and singly mutated at these sites. Dephosphorylation of Syk mutated at these tyrosines reconstituted its capacity to bind to phosphorylated γITAM. B , increased binding of Syk-3F to γPP when its kinase activity was abolished. C , tyrosine 130 had a minor effect on Syk-3F defective γPP binding. Syk tyrosine 130 was substituted with phenylalanine singly (Y130F) or in combination with 3F mutant ( 3F / Y130F ) and their binding to γPP was tested as described above and compared with WT or Syk-3F. Similar results were observed in three independent experiments.
    Figure Legend Snippet: Binding of Syk mutants to phosphorylated γITAM. Cell lysates were prepared in the presence or absence of vanadate from non-stimulated cells 48 h after transfection with the indicated constructs. After incubation for 4 h at 4 °C to allow dephosphorylation of Syk, vanadate was added to all the samples. Lysates were then precipitated with non-phosphorylated (γ YY ) or phosphorylated (γ PP ) ITAM-biotinylated peptides pre-bound to streptavidin-agarose beads. The precipitates were analyzed by immunoblotting with anti-Syk. A , analysis of Syk-3F and singly mutated at these sites. Dephosphorylation of Syk mutated at these tyrosines reconstituted its capacity to bind to phosphorylated γITAM. B , increased binding of Syk-3F to γPP when its kinase activity was abolished. C , tyrosine 130 had a minor effect on Syk-3F defective γPP binding. Syk tyrosine 130 was substituted with phenylalanine singly (Y130F) or in combination with 3F mutant ( 3F / Y130F ) and their binding to γPP was tested as described above and compared with WT or Syk-3F. Similar results were observed in three independent experiments.

    Techniques Used: Binding Assay, Transfection, Construct, Incubation, De-Phosphorylation Assay, Activity Assay, Mutagenesis

    14) Product Images from "RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins"

    Article Title: RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G+-bacterial toxins

    Journal: Biochemical pharmacology

    doi: 10.1016/j.bcp.2016.12.014

    RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P
    Figure Legend Snippet: RhoA is a substrate for S-nitrosylation. (A) HLMVECs were treated with either vehicle or Cys-NO (100 μM) for 30 min, and the S-nitrosylation of proteins was determined by the biotin-switch assay in the presence of ascorbate and trace levels of copper. Biotinylated proteins were concentrated using streptavidin–agarose beads, and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (B) HLMVECs were treated with or without Cys-NO (100 μM) for 30 min, and S-nitrosylated proteins were selected using organomercury columns followed by immunoblotting for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). The relative densitometry of SNO-RhoA vs total-RhoA is expressed as means ± S.E., * P

    Techniques Used: Biotin Switch Assay

    Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P
    Figure Legend Snippet: Mutation of RhoA on C16, 20, 159S reduces the eNOS-dependent S-nitrosylation of RhoA and protects RhoA from the inhibitory effects of NO. (A) COS-7 cells transfected with WT or mutant C16, 20, 159S RhoA constructs were treated with or without Cys-NO (100 μM) for 30 min. Cells were then lysed, the biotin-switch assay performed and biotinylated proteins concentrated using streptavidin agarose. Total S-nitrosylated proteins were identified using an anti-biotin antibody (top panel) and S-nitrosylated RhoA using a RhoA antibody (lower panel). (B) HEK293-eNOS cells were transfected with RhoA WT or the RhoAC16, 20, 159S mutant, and the degree of S-nitrosylation of RhoA was determined using the biotin-switch assay and immunoblotted for RhoA (SNO-RhoA, top panel) versus total RhoA in cell lysates (total RhoA, bottom panel). (C) COS-7 cells were transfected with WT or mutant C16, 20, 159S RhoA and exposed to the indicated concentrations of Cys-NO for 30 min. Cells were then lysed and RhoA activity determined using the G-LISA RhoA activation assay. Data are expressed as means ± S.E., * P

    Techniques Used: Mutagenesis, Transfection, Construct, Biotin Switch Assay, Activity Assay, Activation Assay

    15) Product Images from "Constitutive Endocytosis and Turnover of the Neuronal Glycine Transporter GlyT2 Is Dependent on Ubiquitination of a C-Terminal Lysine Cluster"

    Article Title: Constitutive Endocytosis and Turnover of the Neuronal Glycine Transporter GlyT2 Is Dependent on Ubiquitination of a C-Terminal Lysine Cluster

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058863

    Mutation of each lysine of the GlyT2 C-terminal does not impair GlyT2 constitutive endocytosis. A) Multiple sequence alignment of rat GlyT2 C-terminus region (740–799) from different species was obtained with the CLUSTAL 2.1 multiple sequence alignment method. Identical conserved lysines from different species are shown in red. B-C) MDCK cells expressing wild-type GlyT2 or one of four different point mutants (K751R, K773R, K787R or K791R) were exposed for 30 min to monensin (35 μM) at 37°C or the vehicle alone, fixed with 4% paraformaldehyde, immunostained to visualize GlyT2 and analyzed by confocal microscopy. To simplify the figure, only the wild-type GlyT2 control (Veh) is displayed (all other controls were comparable). Scale bar = 15 μm. C) Representative immunoblot of MDCK cells expressing wild-type GlyT2 or the indicated mutants. Cells were treated with monensin or the vehicle alone, as described above. The cell surface proteins were labeled with sulfo-NHS-SS-biotin and biotinylated proteins were pulled down with streptavidin-agarose beads. GlyT2 expression was analyzed in Western blots and calnexin immunodetection was used as a non-biotinylated protein control. B, biotinylated protein (30 μg); T, total protein (10 μg). D) Densitometric analysis of three independent Western blots as shown in (C), relative to the control values (Veh). E) [ 3 H]-Glycine uptake during 10 minutes was measured in MDCK cells expressing wild-type GlyT2 or the mutants indicated and transport activity is denoted in nmol of glycine/mg of protein. The data represent the means ± SEM and no significant differences respect to vehicle were observed performing ANOVA analysis (with Tukey's post-hoc test).
    Figure Legend Snippet: Mutation of each lysine of the GlyT2 C-terminal does not impair GlyT2 constitutive endocytosis. A) Multiple sequence alignment of rat GlyT2 C-terminus region (740–799) from different species was obtained with the CLUSTAL 2.1 multiple sequence alignment method. Identical conserved lysines from different species are shown in red. B-C) MDCK cells expressing wild-type GlyT2 or one of four different point mutants (K751R, K773R, K787R or K791R) were exposed for 30 min to monensin (35 μM) at 37°C or the vehicle alone, fixed with 4% paraformaldehyde, immunostained to visualize GlyT2 and analyzed by confocal microscopy. To simplify the figure, only the wild-type GlyT2 control (Veh) is displayed (all other controls were comparable). Scale bar = 15 μm. C) Representative immunoblot of MDCK cells expressing wild-type GlyT2 or the indicated mutants. Cells were treated with monensin or the vehicle alone, as described above. The cell surface proteins were labeled with sulfo-NHS-SS-biotin and biotinylated proteins were pulled down with streptavidin-agarose beads. GlyT2 expression was analyzed in Western blots and calnexin immunodetection was used as a non-biotinylated protein control. B, biotinylated protein (30 μg); T, total protein (10 μg). D) Densitometric analysis of three independent Western blots as shown in (C), relative to the control values (Veh). E) [ 3 H]-Glycine uptake during 10 minutes was measured in MDCK cells expressing wild-type GlyT2 or the mutants indicated and transport activity is denoted in nmol of glycine/mg of protein. The data represent the means ± SEM and no significant differences respect to vehicle were observed performing ANOVA analysis (with Tukey's post-hoc test).

    Techniques Used: Mutagenesis, Sequencing, Expressing, Confocal Microscopy, Labeling, Western Blot, Immunodetection, Activity Assay

    The 4KR GlyT2 mutant exhibits impaired endocytosis and lower basal ubiquitination than wild-type GlyT2. A–D) MDCK cells were transfected with wild-type GlyT2 or with 4KR mutant cDNAs (GlyT2 with lysines in positions 751, 773, 787 and 791 mutated to arginines). After 48 h the cells were exposed for 30 min to monensin (35 μM) at 37°C or the vehicle alone, fixed with 4% paraformaldehyde, immunostained to visualize GlyT2 and analyzed by confocal microscopy. Scale bar = 15 μm. Note that endocytosis of the 4KR mutant is blocked in the presence of monensin (D). E) Representative immunoblot of MDCK cells expressing wild-type GlyT2 or the 4KR mutant. Cells were treated for 30 min with the vehicle alone or with monensin (35 μM) at 37°C. Cell surface proteins were labeled with sulfo-NHS-SS-biotin and the biotinylated proteins were pulled down with streptavidin-agarose beads. GlyT2 expression was analyzed in Western blots and calnexin immunodetection was used as a non-biotinylated protein control. B, biotinylated protein (30 μg); T, total protein (10 μg). F) Densitometric analysis of four independent Western blots as in (E) relative to the control values (veh). Data represent means ± SEM. **, significant difference with respect to control, p
    Figure Legend Snippet: The 4KR GlyT2 mutant exhibits impaired endocytosis and lower basal ubiquitination than wild-type GlyT2. A–D) MDCK cells were transfected with wild-type GlyT2 or with 4KR mutant cDNAs (GlyT2 with lysines in positions 751, 773, 787 and 791 mutated to arginines). After 48 h the cells were exposed for 30 min to monensin (35 μM) at 37°C or the vehicle alone, fixed with 4% paraformaldehyde, immunostained to visualize GlyT2 and analyzed by confocal microscopy. Scale bar = 15 μm. Note that endocytosis of the 4KR mutant is blocked in the presence of monensin (D). E) Representative immunoblot of MDCK cells expressing wild-type GlyT2 or the 4KR mutant. Cells were treated for 30 min with the vehicle alone or with monensin (35 μM) at 37°C. Cell surface proteins were labeled with sulfo-NHS-SS-biotin and the biotinylated proteins were pulled down with streptavidin-agarose beads. GlyT2 expression was analyzed in Western blots and calnexin immunodetection was used as a non-biotinylated protein control. B, biotinylated protein (30 μg); T, total protein (10 μg). F) Densitometric analysis of four independent Western blots as in (E) relative to the control values (veh). Data represent means ± SEM. **, significant difference with respect to control, p

    Techniques Used: Mutagenesis, Transfection, Confocal Microscopy, Expressing, Labeling, Western Blot, Immunodetection

    UCHL1 inhibition impairs Glyt2 constitutive endocytosis in neurons. A) Representative immunoblot of brainstem and spinal cord primary neurons. Cells were pretreated for 2 h with vehicle (DMSO) or LDN -57444 (UCHL1 inhibitor: 10 μM) and were then exposed to monensin (35 μM, 30 min) or the vehicle alone (EtOH), in the presence or absence of UCHL1. Cell surface proteins were labeled with sulfo-NHS-SS-biotin and the biotinylated proteins were pulled down with streptavidin-agarose beads. GlyT2 expression was analyzed in Western blots using calnexin immunodetection as a control of intracellular non-biotinylated protein. B, biotinylated protein (30 μg); T, total protein (10 μg). B) Densitometric analysis of four independent Western blots as in (A) relative to the control values (Veh). Data represent the means ± SEM. **, significant difference with respect to control; p
    Figure Legend Snippet: UCHL1 inhibition impairs Glyt2 constitutive endocytosis in neurons. A) Representative immunoblot of brainstem and spinal cord primary neurons. Cells were pretreated for 2 h with vehicle (DMSO) or LDN -57444 (UCHL1 inhibitor: 10 μM) and were then exposed to monensin (35 μM, 30 min) or the vehicle alone (EtOH), in the presence or absence of UCHL1. Cell surface proteins were labeled with sulfo-NHS-SS-biotin and the biotinylated proteins were pulled down with streptavidin-agarose beads. GlyT2 expression was analyzed in Western blots using calnexin immunodetection as a control of intracellular non-biotinylated protein. B, biotinylated protein (30 μg); T, total protein (10 μg). B) Densitometric analysis of four independent Western blots as in (A) relative to the control values (Veh). Data represent the means ± SEM. **, significant difference with respect to control; p

    Techniques Used: Inhibition, Labeling, Expressing, Western Blot, Immunodetection

    16) Product Images from "Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers"

    Article Title: Transcriptional coactivator CBP upregulates hTERT expression and tumor growth and predicts poor prognosis in human lung cancers

    Journal: Oncotarget

    doi:

    The interaction of CBP with Sp1 and AP-2 and the acetylationt of Sp1 by CBP in lung cancer cells (A) The nuclear extracts of human lung normal and cancer cells were prepared for immunoprecipitation using an antibody against Sp1 or AP-2β and then evaluated by immunoblot using antibody against CBP. (B) Human lung cancer H1299 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of CBP with Sp1 or AP-2β were examined by confocal microscopy analysis with a confocal microscope. More than 100 cells were inspected per experiment, and cells with typical morphology were presented. (C) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done. Sp1 was tested in the pulled down proteins by immunoblot using antibody against Sp1. (D) Chromatin immunoprecipitation assays were done using antibody against Sp1. PCR products of hTERT promoter (-378 to +60) were separated on 1% agarose gels. The last lane represents the IgG control. (E) Immunoprecipitation was performed using antibody against Sp1. The acetylated Sp1 was determined by immunoblot using the antibody against acetylation. (F) Immunoprecipitation was performed in human lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or inhibitor using antibody against Sp1. The acetylated Sp1 was tested by immunoblot using antibody against acetylation. (G) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done in lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or CBP-specific inhibitor. The level of Sp1 in the pulled down proteins was determined by immunoblot. Densitometric analysis was used to analyze quantitatively the binding activity and acetylation level of Sp1 proteins.
    Figure Legend Snippet: The interaction of CBP with Sp1 and AP-2 and the acetylationt of Sp1 by CBP in lung cancer cells (A) The nuclear extracts of human lung normal and cancer cells were prepared for immunoprecipitation using an antibody against Sp1 or AP-2β and then evaluated by immunoblot using antibody against CBP. (B) Human lung cancer H1299 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of CBP with Sp1 or AP-2β were examined by confocal microscopy analysis with a confocal microscope. More than 100 cells were inspected per experiment, and cells with typical morphology were presented. (C) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done. Sp1 was tested in the pulled down proteins by immunoblot using antibody against Sp1. (D) Chromatin immunoprecipitation assays were done using antibody against Sp1. PCR products of hTERT promoter (-378 to +60) were separated on 1% agarose gels. The last lane represents the IgG control. (E) Immunoprecipitation was performed using antibody against Sp1. The acetylated Sp1 was determined by immunoblot using the antibody against acetylation. (F) Immunoprecipitation was performed in human lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or inhibitor using antibody against Sp1. The acetylated Sp1 was tested by immunoblot using antibody against acetylation. (G) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done in lung cancer cells (H1299) treated by non-specific siRNA or CBP specific siRNA or CBP-specific inhibitor. The level of Sp1 in the pulled down proteins was determined by immunoblot. Densitometric analysis was used to analyze quantitatively the binding activity and acetylation level of Sp1 proteins.

    Techniques Used: Immunoprecipitation, Confocal Microscopy, Microscopy, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay, Activity Assay

    Identification of CBP as a hTERT promoter-binding protein in lung cancer cells (A) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done in human normal lung cells and lung cancer cells. The pulled down proteins were tested by immunoblot using antibodies against CBP. (B) Chromatin immunoprecipitation assay was done with normal lung cells and lung adenocarcinoma cells using antibodies against CBP. PCR products were separated on 1% agarose gels. The last lane represents the IgG control. The displayed gels were representative of 2-3 independent experiments. Densitometric analysis was used to analyze quantitatively the binding activity of CBP protein on hTERT promoter.
    Figure Legend Snippet: Identification of CBP as a hTERT promoter-binding protein in lung cancer cells (A) Streptavidin-agarose bead pulldown assay with hTERT promoter (-378 to +60) as probes was done in human normal lung cells and lung cancer cells. The pulled down proteins were tested by immunoblot using antibodies against CBP. (B) Chromatin immunoprecipitation assay was done with normal lung cells and lung adenocarcinoma cells using antibodies against CBP. PCR products were separated on 1% agarose gels. The last lane represents the IgG control. The displayed gels were representative of 2-3 independent experiments. Densitometric analysis was used to analyze quantitatively the binding activity of CBP protein on hTERT promoter.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Activity Assay

    17) Product Images from "Hsp70 Chaperones and Type I PRMTs Are Sequestered at Intranuclear Inclusions Caused by Polyalanine Expansions in PABPN1"

    Article Title: Hsp70 Chaperones and Type I PRMTs Are Sequestered at Intranuclear Inclusions Caused by Polyalanine Expansions in PABPN1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006418

    Identification of proteins that associate preferentially with expanded PABPN1. (A) Purification strategy. Recombinant, His-tagged PABPN1 containing either a normal homopolymer of 10 alanine residues or an expanded tract of 17 alanine residues was expressed in baculovirus system and purified by nickel affinity. Normal and expanded PABPN1 were then biotinylated, immobilized to streptavidin-agarose beads and incubated with RNase-treated extracts from undifferentiated C2 (myoblasts) and differentiated C2 (myotubes) cells. (B) Coomassie-stained SDS polyacrylamide gel of purified recombinant HIS-PABPN1 used for pull-down experiments. (C) Bound proteins were eluted, separated by 10% SDS-PAGE, and detected by silver staining. The gel bands that stained with higher intensity in the lanes corresponding to expanded PABPN1 were excised and proteins identified by mass spectrometry. The identity of the bands is indicated. As a control, the same amount of extract was incubated with beads devoid of any immobilized protein (control lanes). (D) Proteins bound to normal and expanded PABPN1 were eluted, separated by 10% SDS-PAGE, blotted to nitrocellulose, and probed with the antibodies against the indicated proteins. Total protein from C2 cell extract (input) was run in parallel. Lower panel shows the corresponding loading control for wt- and expanded PABPN1 proteins in cell free extracts obtained from myoblasts and myotubes.
    Figure Legend Snippet: Identification of proteins that associate preferentially with expanded PABPN1. (A) Purification strategy. Recombinant, His-tagged PABPN1 containing either a normal homopolymer of 10 alanine residues or an expanded tract of 17 alanine residues was expressed in baculovirus system and purified by nickel affinity. Normal and expanded PABPN1 were then biotinylated, immobilized to streptavidin-agarose beads and incubated with RNase-treated extracts from undifferentiated C2 (myoblasts) and differentiated C2 (myotubes) cells. (B) Coomassie-stained SDS polyacrylamide gel of purified recombinant HIS-PABPN1 used for pull-down experiments. (C) Bound proteins were eluted, separated by 10% SDS-PAGE, and detected by silver staining. The gel bands that stained with higher intensity in the lanes corresponding to expanded PABPN1 were excised and proteins identified by mass spectrometry. The identity of the bands is indicated. As a control, the same amount of extract was incubated with beads devoid of any immobilized protein (control lanes). (D) Proteins bound to normal and expanded PABPN1 were eluted, separated by 10% SDS-PAGE, blotted to nitrocellulose, and probed with the antibodies against the indicated proteins. Total protein from C2 cell extract (input) was run in parallel. Lower panel shows the corresponding loading control for wt- and expanded PABPN1 proteins in cell free extracts obtained from myoblasts and myotubes.

    Techniques Used: Purification, Recombinant, Incubation, Staining, SDS Page, Silver Staining, Mass Spectrometry

    18) Product Images from "Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import"

    Article Title: Identification of Protein Cofactors Necessary for Sequence-specific Plasmid DNA Nuclear Import

    Journal: Molecular Therapy

    doi: 10.1038/mt.2009.127

    Precipitation of the protein–pDNA import complex in living cells. ( a ) Human smooth muscle cells were electroporated with plasmid DNA (pDNA) that was hybridized to a biotinylated peptide nucleic acid clamp. At 60 and 240 minutes post-transfection, cells were treated with formaldehyde to cross-link proteins bound to the pDNA, lysed and precipitated using streptavidin-agarose. Unbound fractions (Sup) and precipitated fractions (Pel) were probed for putative members of the import complex using western blots. ( b ) To verify equal levels of transfection efficiency, crude lysates from transfected cells were Slot blotted onto membranes, UV cross-linked and probed for biotinylated pDNA using horseradish peroxidase–streptavidin. DTS, DNA nuclear targeting sequence; SMGA, smooth muscle gamma actin; SV40, simian virus 40.
    Figure Legend Snippet: Precipitation of the protein–pDNA import complex in living cells. ( a ) Human smooth muscle cells were electroporated with plasmid DNA (pDNA) that was hybridized to a biotinylated peptide nucleic acid clamp. At 60 and 240 minutes post-transfection, cells were treated with formaldehyde to cross-link proteins bound to the pDNA, lysed and precipitated using streptavidin-agarose. Unbound fractions (Sup) and precipitated fractions (Pel) were probed for putative members of the import complex using western blots. ( b ) To verify equal levels of transfection efficiency, crude lysates from transfected cells were Slot blotted onto membranes, UV cross-linked and probed for biotinylated pDNA using horseradish peroxidase–streptavidin. DTS, DNA nuclear targeting sequence; SMGA, smooth muscle gamma actin; SV40, simian virus 40.

    Techniques Used: Plasmid Preparation, Transfection, Western Blot, Sequencing

    19) Product Images from "TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1"

    Article Title: TOPLESS mediates brassinosteroid-induced transcriptional repression through interaction with BZR1

    Journal: Nature communications

    doi: 10.1038/ncomms5140

    BZR1 interacts with TOPLESS through the EAR motif (a) A yeast two-hybrid assay shows the interaction between BZR1 and TPL. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 50 mM 3AT medium. (b) Mutation in the EAR motif abolishes the BZR1-TPL interaction. Core Leu residues of the EAR motif were substituted to Ala. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 1 mM 3AT medium. (c) BZR1 interacts with TPL and other TPRs. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 5 mM 3AT medium. TPL-N: N-terminal domain of TPL (1–344), TPR1-N : 1–343, TPR2-N : 1–335, TPR3-N : 1–335 and TPR4-N : 1–344. (d) BZR1 interacts with TPL through EAR motif in vivo . Arabidopsis mesophyll protoplasts were transfected with TPL-Myc together with bzr1-1D-GFP or bzr1-1DΔEAR-GFP , and the extracted proteins were immunoprecipitated by anti-GFP antibody. Gel blots were probed with anti-Myc or anti-GFP antibody. (e) TPL binds to DWF4 promoter through BZR1. GST-TPL-N (amino acids 1–344) was incubated with a biotin-labelled DWF4 promoter DNA (400 bps) together with MBP or MBP-BZR1 and pulled down with streptavidin agarose beads. Gel blots were probed with anti-GST or anti-MBP antibody. The full scans of the gel blots (d,e) are shown in Supplementary Fig. 4 . (f) The TPL DNA-binding on the promoters of CPD and DWF4 are enhanced by BR treatment. TPL DNA-binding was determined by ChIP assay using TPLp::TPL-HA transgenic plants. One-week seedlings grown on the medium containing 2 µM PPZ were treated with mock (M) or 100 nM BL for 4 hr before crosslinking. Error bars indicate the s.d. ( n =3) and **: P
    Figure Legend Snippet: BZR1 interacts with TOPLESS through the EAR motif (a) A yeast two-hybrid assay shows the interaction between BZR1 and TPL. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 50 mM 3AT medium. (b) Mutation in the EAR motif abolishes the BZR1-TPL interaction. Core Leu residues of the EAR motif were substituted to Ala. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 1 mM 3AT medium. (c) BZR1 interacts with TPL and other TPRs. Yeast clones were grown on the synthetic dropout (+His) or synthetic dropout without histidine (−His) plus 5 mM 3AT medium. TPL-N: N-terminal domain of TPL (1–344), TPR1-N : 1–343, TPR2-N : 1–335, TPR3-N : 1–335 and TPR4-N : 1–344. (d) BZR1 interacts with TPL through EAR motif in vivo . Arabidopsis mesophyll protoplasts were transfected with TPL-Myc together with bzr1-1D-GFP or bzr1-1DΔEAR-GFP , and the extracted proteins were immunoprecipitated by anti-GFP antibody. Gel blots were probed with anti-Myc or anti-GFP antibody. (e) TPL binds to DWF4 promoter through BZR1. GST-TPL-N (amino acids 1–344) was incubated with a biotin-labelled DWF4 promoter DNA (400 bps) together with MBP or MBP-BZR1 and pulled down with streptavidin agarose beads. Gel blots were probed with anti-GST or anti-MBP antibody. The full scans of the gel blots (d,e) are shown in Supplementary Fig. 4 . (f) The TPL DNA-binding on the promoters of CPD and DWF4 are enhanced by BR treatment. TPL DNA-binding was determined by ChIP assay using TPLp::TPL-HA transgenic plants. One-week seedlings grown on the medium containing 2 µM PPZ were treated with mock (M) or 100 nM BL for 4 hr before crosslinking. Error bars indicate the s.d. ( n =3) and **: P

    Techniques Used: Y2H Assay, Clone Assay, Mutagenesis, In Vivo, Transfection, Immunoprecipitation, Incubation, Binding Assay, Chromatin Immunoprecipitation, Transgenic Assay

    20) Product Images from "U7 snRNP is recruited to histone pre-mRNA in a FLASH-dependent manner by two separate regions of the stem–loop binding protein"

    Article Title: U7 snRNP is recruited to histone pre-mRNA in a FLASH-dependent manner by two separate regions of the stem–loop binding protein

    Journal: RNA

    doi: 10.1261/rna.060806.117

    Characterization of mouse processing complexes. ( A ) Processing complexes were formed on 5′Biot-mH2a/5m pre-mRNA in a mouse nuclear extract in the absence or presence of N-terminal human FLASH (FLASH/N, amino acids 1–138) and/or SL RNA, as indicated. The bound proteins were separated in a 4%–12% SDS/polyacrylamide gel and detected by Western blotting. Lane 1 contains 2.5% of the input nuclear extract that lacks recombinant FLASH. The two bands detected in this lane by the antibody against the N-terminal FLASH (indicated with asterisks) likely represent cross-reactive proteins. Note that SLBP is undetectable in this amount of input. ( B ) A mouse nuclear extract containing human FLASH/N was used to assemble processing complexes on 5′Biot-mH2a/5m pre-mRNA either in the absence (lane 2 ) or in the presence (lane 3 ) of antiU7 oligonucleotide that blocks the 5′ end of U7 snRNA. In lane 1 , the pre-mRNA was omitted. Proteins purified on streptavidin beads were separated in a 4%–12% SDS/polyacrylamide gel and visualized with silver ( left ). Small sections (bands A – F ) were excised from the same areas of lanes 2 and 3 and analyzed by mass spectrometry to identify their proteome ( right ). Proteins with the top three scores in each band, with the exception of band B where only two proteins were identified, are listed in the table. ( C ) Processing complexes were formed on 5′Biot-mH2a/5m pre-mRNA in a mouse nuclear extract either lacking (lane 1 ) or containing human FLASH/N (lanes 2 , 3 ). In lane 3 , binding of U7 snRNP to 5′Biot-mH2a/5m pre-mRNA was prevented by an antiU7 oligonucleotide. Proteins purified on streptavidin beads were separated in a 4%–12% SDS/polyacrylamide gel and visualized by silver staining.
    Figure Legend Snippet: Characterization of mouse processing complexes. ( A ) Processing complexes were formed on 5′Biot-mH2a/5m pre-mRNA in a mouse nuclear extract in the absence or presence of N-terminal human FLASH (FLASH/N, amino acids 1–138) and/or SL RNA, as indicated. The bound proteins were separated in a 4%–12% SDS/polyacrylamide gel and detected by Western blotting. Lane 1 contains 2.5% of the input nuclear extract that lacks recombinant FLASH. The two bands detected in this lane by the antibody against the N-terminal FLASH (indicated with asterisks) likely represent cross-reactive proteins. Note that SLBP is undetectable in this amount of input. ( B ) A mouse nuclear extract containing human FLASH/N was used to assemble processing complexes on 5′Biot-mH2a/5m pre-mRNA either in the absence (lane 2 ) or in the presence (lane 3 ) of antiU7 oligonucleotide that blocks the 5′ end of U7 snRNA. In lane 1 , the pre-mRNA was omitted. Proteins purified on streptavidin beads were separated in a 4%–12% SDS/polyacrylamide gel and visualized with silver ( left ). Small sections (bands A – F ) were excised from the same areas of lanes 2 and 3 and analyzed by mass spectrometry to identify their proteome ( right ). Proteins with the top three scores in each band, with the exception of band B where only two proteins were identified, are listed in the table. ( C ) Processing complexes were formed on 5′Biot-mH2a/5m pre-mRNA in a mouse nuclear extract either lacking (lane 1 ) or containing human FLASH/N (lanes 2 , 3 ). In lane 3 , binding of U7 snRNP to 5′Biot-mH2a/5m pre-mRNA was prevented by an antiU7 oligonucleotide. Proteins purified on streptavidin beads were separated in a 4%–12% SDS/polyacrylamide gel and visualized by silver staining.

    Techniques Used: Western Blot, Recombinant, Purification, Mass Spectrometry, Binding Assay, Silver Staining

    Drosophila SLBP promotes the recruitment of U7 snRNP to histone pre-mRNA. ( A ) Three potential forms of U7 snRNP in nuclear extracts from animal cells. The core U7 snRNP consists of the Sm ring and U7 snRNA. Lsm11 interacts with FLASH to generate the FLASH-bound form of U7 snRNP. This step may be cell cycle regulated, occurring during G1/S transition. FLASH and Lsm11 act together to recruit a number of polyadenylation factors, the histone pre-mRNA cleavage complex (HCC), giving rise to the holo-U7 snRNP. Only the key HCC subunits are shown and their arrangement in the complex is arbitrary. ( B ) Schematic representation of the 3′Biot-dH3 pre-mRNA used to assemble and purify Drosophila processing complexes. The 65-histone pre-mRNA fragment contains a stem–loop and HDE that bind SLBP and U7 snRNP, respectively (see Materials and Methods for the sequence). Two 2′ O -methyl modifications were placed downstream from the cleavage site (Endo) to block potential 5′–3′ exonuclease activity of CPSF73 after the cleavage step (5′ Exo). A biotin tag is covalently attached to the 3′ end, leaving the 5′ end for labeling with 32 P, when desired. ( C ) Proteins bound to 3′Biot-dH3 pre-mRNA in a Drosophila nuclear extract in the absence (lane 2 ) or in the presence of indicated competitors (lanes 3 , 4 ) were separated in a 4%–12% SDS/polyacrylamide gel and analyzed by Western blotting using antibodies against known processing factors. Lane 1 contains 2.5% of the input nuclear extract used for formation of the processing complexes. Note that SLBP is limiting in Kc nuclear extracts and undetectable in this amount of the input. Size markers in kDa are shown to the right . ( D , E ) 3′Biot-dH3 pre-mRNA labeled at the 5′ end was incubated with a Drosophila nuclear extract for 15 min either on ice or at room temperature to prevent or allow cleavage, respectively. RNA was isolated from a small portion of each reaction and separated in an 8% denaturing polyacrylamide gel to analyze the extent of cleavage (panel D ). Processing complexes were purified on streptavidin beads from the remaining part of each reaction, separated in a 4%–12% SDS/polyacrylamide gel and analyzed for the presence of selected processing factors by Western blotting (panel E ). Lane 1 in panel E contains 2.5% of the input nuclear extract. Note that this amount is insufficient to detect SLBP.
    Figure Legend Snippet: Drosophila SLBP promotes the recruitment of U7 snRNP to histone pre-mRNA. ( A ) Three potential forms of U7 snRNP in nuclear extracts from animal cells. The core U7 snRNP consists of the Sm ring and U7 snRNA. Lsm11 interacts with FLASH to generate the FLASH-bound form of U7 snRNP. This step may be cell cycle regulated, occurring during G1/S transition. FLASH and Lsm11 act together to recruit a number of polyadenylation factors, the histone pre-mRNA cleavage complex (HCC), giving rise to the holo-U7 snRNP. Only the key HCC subunits are shown and their arrangement in the complex is arbitrary. ( B ) Schematic representation of the 3′Biot-dH3 pre-mRNA used to assemble and purify Drosophila processing complexes. The 65-histone pre-mRNA fragment contains a stem–loop and HDE that bind SLBP and U7 snRNP, respectively (see Materials and Methods for the sequence). Two 2′ O -methyl modifications were placed downstream from the cleavage site (Endo) to block potential 5′–3′ exonuclease activity of CPSF73 after the cleavage step (5′ Exo). A biotin tag is covalently attached to the 3′ end, leaving the 5′ end for labeling with 32 P, when desired. ( C ) Proteins bound to 3′Biot-dH3 pre-mRNA in a Drosophila nuclear extract in the absence (lane 2 ) or in the presence of indicated competitors (lanes 3 , 4 ) were separated in a 4%–12% SDS/polyacrylamide gel and analyzed by Western blotting using antibodies against known processing factors. Lane 1 contains 2.5% of the input nuclear extract used for formation of the processing complexes. Note that SLBP is limiting in Kc nuclear extracts and undetectable in this amount of the input. Size markers in kDa are shown to the right . ( D , E ) 3′Biot-dH3 pre-mRNA labeled at the 5′ end was incubated with a Drosophila nuclear extract for 15 min either on ice or at room temperature to prevent or allow cleavage, respectively. RNA was isolated from a small portion of each reaction and separated in an 8% denaturing polyacrylamide gel to analyze the extent of cleavage (panel D ). Processing complexes were purified on streptavidin beads from the remaining part of each reaction, separated in a 4%–12% SDS/polyacrylamide gel and analyzed for the presence of selected processing factors by Western blotting (panel E ). Lane 1 in panel E contains 2.5% of the input nuclear extract. Note that this amount is insufficient to detect SLBP.

    Techniques Used: Activated Clotting Time Assay, Sequencing, Blocking Assay, Activity Assay, Labeling, Western Blot, Incubation, Isolation, Purification

    The recruitment of U7 snRNP to histone pre-mRNA by Drosophila SLBP. ( A ) Schematic representation of the experiment to test the activity of recombinant Drosophila SLBPs in stimulating the recruitment of U7 snRNP to 3′Biot-dH3 histone pre-mRNA in a Drosophila nuclear extract containing stem–loop RNA (SL). Histone pre-mRNA is quantitatively prebound to an excess of recombinant SLBP, and the complex is purified on streptavidin (SA) agarose beads via the 3′ biotin tag (step 1). Drosophila nuclear extract is briefly mixed with excess SL RNA to sequester endogenous SLBP and incubated with SA beads containing 3′Biot-dH3 pre-mRNA bound to recombinant SLBP (step 2). Complexes immobilized on SA beads are extensively washed and analyzed by Western blotting for the presence of processing factors using specific antibodies (step 3). ( B ) Processing complexes were formed in the absence of Drosophila SLBP (lane 2 ) or in the presence of various recombinant SLBPs prebound to histone pre-mRNA, as indicated at the top of lanes 3 – 6 , and analyzed for the presence of selected processing factors by Western blotting. A fraction of the nuclear extract used in the experiment (2.5%) is shown in lane 1 . Note that this input lane and lane 2 contain no Flag-tagged SLBP. ( C ) Processing complexes were formed in the presence of prebound WT SLBP (lanes 2 , 3 ) or 4S-4A mutant SLBP (lanes 4 , 5 ), with the proteins being either in a native form (lanes 2 , 4 ) or pretreated with calf intestinal phosphatase (CIP) to remove phosphate groups (lanes 3 , 5 ). Processing complexes formed in the absence of recombinant SLBP are shown in lane 1 . ( D ) Processing complexes were formed in the presence of prebound WT SLBP (lane 2 ) or 4S-4E mutant SLBP that was either untreated (lane 3 ) or pretreated with CIP to remove phosphate groups (lane 4 ). Processing complexes formed in the absence of recombinant SLBP are shown in lane 1 .
    Figure Legend Snippet: The recruitment of U7 snRNP to histone pre-mRNA by Drosophila SLBP. ( A ) Schematic representation of the experiment to test the activity of recombinant Drosophila SLBPs in stimulating the recruitment of U7 snRNP to 3′Biot-dH3 histone pre-mRNA in a Drosophila nuclear extract containing stem–loop RNA (SL). Histone pre-mRNA is quantitatively prebound to an excess of recombinant SLBP, and the complex is purified on streptavidin (SA) agarose beads via the 3′ biotin tag (step 1). Drosophila nuclear extract is briefly mixed with excess SL RNA to sequester endogenous SLBP and incubated with SA beads containing 3′Biot-dH3 pre-mRNA bound to recombinant SLBP (step 2). Complexes immobilized on SA beads are extensively washed and analyzed by Western blotting for the presence of processing factors using specific antibodies (step 3). ( B ) Processing complexes were formed in the absence of Drosophila SLBP (lane 2 ) or in the presence of various recombinant SLBPs prebound to histone pre-mRNA, as indicated at the top of lanes 3 – 6 , and analyzed for the presence of selected processing factors by Western blotting. A fraction of the nuclear extract used in the experiment (2.5%) is shown in lane 1 . Note that this input lane and lane 2 contain no Flag-tagged SLBP. ( C ) Processing complexes were formed in the presence of prebound WT SLBP (lanes 2 , 3 ) or 4S-4A mutant SLBP (lanes 4 , 5 ), with the proteins being either in a native form (lanes 2 , 4 ) or pretreated with calf intestinal phosphatase (CIP) to remove phosphate groups (lanes 3 , 5 ). Processing complexes formed in the absence of recombinant SLBP are shown in lane 1 . ( D ) Processing complexes were formed in the presence of prebound WT SLBP (lane 2 ) or 4S-4E mutant SLBP that was either untreated (lane 3 ) or pretreated with CIP to remove phosphate groups (lane 4 ). Processing complexes formed in the absence of recombinant SLBP are shown in lane 1 .

    Techniques Used: Activity Assay, Recombinant, Purification, Incubation, Western Blot, Mutagenesis

    21) Product Images from "Essential interaction between the fission yeast DNA polymerase ? subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal p21Cip1-like PCNA binding motif"

    Article Title: Essential interaction between the fission yeast DNA polymerase ? subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal p21Cip1-like PCNA binding motif

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.5.1108

    Fig. 4. In vitro Pcn1 binding assay using biotinylated PP–Cdc27 fusion protein (PP–Cdc27). Upper part: schematic representation of PP–Cdc27 showing the location of the biotinylated lysine residue and, at the C–terminus, residues corresponding to 353–372 in Cdc27. Lower part: following mixing of the PP–Cdc27 protein with recombinant Pcn1, proteins binding to Pcn1 were isolated by Ni–NTA affinity chromatography and subjected to SDS–PAGE. The bound proteins were then transferred to a PVDF membrane and probed using streptavidin-labelled alkaline phosphatase to detect the presence of the biotinylated PP–Cdc27 protein. Retention of the PP–Cdc27 protein was dependent upon the presence of Pcn1 (lane 2). See the text for details.
    Figure Legend Snippet: Fig. 4. In vitro Pcn1 binding assay using biotinylated PP–Cdc27 fusion protein (PP–Cdc27). Upper part: schematic representation of PP–Cdc27 showing the location of the biotinylated lysine residue and, at the C–terminus, residues corresponding to 353–372 in Cdc27. Lower part: following mixing of the PP–Cdc27 protein with recombinant Pcn1, proteins binding to Pcn1 were isolated by Ni–NTA affinity chromatography and subjected to SDS–PAGE. The bound proteins were then transferred to a PVDF membrane and probed using streptavidin-labelled alkaline phosphatase to detect the presence of the biotinylated PP–Cdc27 protein. Retention of the PP–Cdc27 protein was dependent upon the presence of Pcn1 (lane 2). See the text for details.

    Techniques Used: In Vitro, Binding Assay, Recombinant, Isolation, Affinity Chromatography, SDS Page

    Fig. 5. Peptide–Pcn1 interactions. ( A ) Peptides were conjugated to streptavidin–agarose beads and incubated with S.pombe cell extracts. Following recovery and extensive washing of the beads, the bound PCNA was analysed by SDS–PAGE followed by Western blot analysis with the monoclonal anti-PCNA antibody PC10. The peptides used are described in Materials and methods, and represent previously described PCNA binding peptides from human p21 Cip1 (p21), and C–terminal 20 amino acid sequences derived from Cdc27 (Cdc27) and Pol32 (Pol32). In each case, peptides were also tested in which the conserved glutamine was substituted with alanine (peptide-A). A peptide of unrelated sequence was used as a control for non-specific binding. ( B ) The ability of immobilized peptides to bind to PCNA was tested in the presence of either the p21 Cip1 -derived peptide (+), an unrelated control peptide (c) or the solvent DMSO (–). These were added to diluted S.pombe cell extracts before incubation with the immobilized, biotinylated peptides. The competitor p21 Cip1 ).
    Figure Legend Snippet: Fig. 5. Peptide–Pcn1 interactions. ( A ) Peptides were conjugated to streptavidin–agarose beads and incubated with S.pombe cell extracts. Following recovery and extensive washing of the beads, the bound PCNA was analysed by SDS–PAGE followed by Western blot analysis with the monoclonal anti-PCNA antibody PC10. The peptides used are described in Materials and methods, and represent previously described PCNA binding peptides from human p21 Cip1 (p21), and C–terminal 20 amino acid sequences derived from Cdc27 (Cdc27) and Pol32 (Pol32). In each case, peptides were also tested in which the conserved glutamine was substituted with alanine (peptide-A). A peptide of unrelated sequence was used as a control for non-specific binding. ( B ) The ability of immobilized peptides to bind to PCNA was tested in the presence of either the p21 Cip1 -derived peptide (+), an unrelated control peptide (c) or the solvent DMSO (–). These were added to diluted S.pombe cell extracts before incubation with the immobilized, biotinylated peptides. The competitor p21 Cip1 ).

    Techniques Used: Incubation, SDS Page, Western Blot, Binding Assay, Derivative Assay, Sequencing

    22) Product Images from "Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1"

    Article Title: Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkg817

    CDC5L peptides associate with PLRG1 in HeLa nuclear extract. CDC5L peptides were used in pull-down assays on streptavidin–agarose beads and the co-precipitated protein transferred to the nitrocellulose membranes was probed with anti-PLRG1 antibodies. Protein bands were then revealed by enhanced chemiluminescence (ECL). ( A ) Lane 1, a positive control containing HeLa nuclear extract; lanes 2 and 3, control pull-downs using beads only and CD-R24 peptide, respectively; lanes 4–6, protein from pull-down assays using the CD24-1, CD24-2 and CD24-3 peptides, respectively. ( B ) Pull-down assays using 12mer CDC5L peptides. Lane 1, a control containing HeLa nuclear extract; lane 2, a control pull-down assay using the CD-R12 peptide; lanes 3–5, protein from pull-down assays using the CD12-1, CD12-2 and CD12-3 peptides, respectively. ( C ) Binding of CDC5L peptides to PLRG1 in nuclear extract does not disrupt the CDC5L–PLRG1 interaction. Pull-down assays were performed as above using streptavidin–agarose beads except that the blots were probed with a buffer containing both anti-PLRG1 and anti-CDC5L antibodies. Lane 1, the positive control (HeLa nuclear extract); lanes 2 and 3, control pull-downs using the CD-R12 and CD-R24 peptides, respectively; lanes 4 and 5, pull-downs performed using the CD12-3 and CD24-1 peptides, respectively. The arrowheads on the right of the figure point to the bands representing PLRG1 or CDC5L on the nitrocellulose membrane.
    Figure Legend Snippet: CDC5L peptides associate with PLRG1 in HeLa nuclear extract. CDC5L peptides were used in pull-down assays on streptavidin–agarose beads and the co-precipitated protein transferred to the nitrocellulose membranes was probed with anti-PLRG1 antibodies. Protein bands were then revealed by enhanced chemiluminescence (ECL). ( A ) Lane 1, a positive control containing HeLa nuclear extract; lanes 2 and 3, control pull-downs using beads only and CD-R24 peptide, respectively; lanes 4–6, protein from pull-down assays using the CD24-1, CD24-2 and CD24-3 peptides, respectively. ( B ) Pull-down assays using 12mer CDC5L peptides. Lane 1, a control containing HeLa nuclear extract; lane 2, a control pull-down assay using the CD-R12 peptide; lanes 3–5, protein from pull-down assays using the CD12-1, CD12-2 and CD12-3 peptides, respectively. ( C ) Binding of CDC5L peptides to PLRG1 in nuclear extract does not disrupt the CDC5L–PLRG1 interaction. Pull-down assays were performed as above using streptavidin–agarose beads except that the blots were probed with a buffer containing both anti-PLRG1 and anti-CDC5L antibodies. Lane 1, the positive control (HeLa nuclear extract); lanes 2 and 3, control pull-downs using the CD-R12 and CD-R24 peptides, respectively; lanes 4 and 5, pull-downs performed using the CD12-3 and CD24-1 peptides, respectively. The arrowheads on the right of the figure point to the bands representing PLRG1 or CDC5L on the nitrocellulose membrane.

    Techniques Used: Positive Control, Pull Down Assay, Binding Assay

    PLRG1 peptides will interact with CDC5L in nuclear extract and inhibit pre-mRNA splicing. ( A ) Design of peptides from sequences in the CDC5L binding region of PLRG1. The arrows indicate the sequences of the peptides synthesised. ( B ) Autoradiograph of a splicing gel from an experiment to determine the effect of 24mer–30mer peptides spanning the highly conserved WD40 sequences on splicing. Approximately 7–20 nmol peptide were added to the splicing reactions (lanes 4–12). Lane 1 contained the input pre-mRNA. CTRL1 is a control splicing reaction without peptide. CTRL2 is a control reaction containing 20 nmol control peptide HC-2 derived from another spliceosomal protein HCF-1 that has not been detected in complexes containing CDC5L and PLRG1. The symbols on the right of the panel represent the input RNA, splicing intermediates and products. ( C ) Autoradiograph of a splicing gel from an experiment to determine the effect of overlapping 15mer peptides spanning the PL30-3 sequence on splicing. Similar amounts of peptide were added (lanes 4–12) to the splicing reactions as in (B). The lanes marked CTRL1 and CTRL2 contained splicing reactions treated in a similar way to lanes with the same names in (B). ( D ) Peptides containing the same amino acids as PL15-3 and PL30-3 in a scrambled sequence do not inhibit splicing. Lane 1, CTRL1 is the control reaction without peptide; lane 2, ∼20 nmol PL30-3; lanes 3–5, 7–20 nmol PL-SB15; lanes 6–8, 7–20 nmol PL-SB30 peptide. ( E ) Pull-down of CDC5L onto streptavidin–agarose beads from HeLa nuclear extract using PLRG1 peptides. Lanes 4–9, pull-down assays with the corresponding peptides (used in marking each lane). CTRL1 did not contain any peptides whereas CTRL2 contained a control peptide that does not inhibit splicing. The blot was probed with a buffer containing both anti-CDC5L and anti-PLRG1 antibodies. The arrows on the right of the panel show the positions of the CDC5L and PLRG1 proteins.
    Figure Legend Snippet: PLRG1 peptides will interact with CDC5L in nuclear extract and inhibit pre-mRNA splicing. ( A ) Design of peptides from sequences in the CDC5L binding region of PLRG1. The arrows indicate the sequences of the peptides synthesised. ( B ) Autoradiograph of a splicing gel from an experiment to determine the effect of 24mer–30mer peptides spanning the highly conserved WD40 sequences on splicing. Approximately 7–20 nmol peptide were added to the splicing reactions (lanes 4–12). Lane 1 contained the input pre-mRNA. CTRL1 is a control splicing reaction without peptide. CTRL2 is a control reaction containing 20 nmol control peptide HC-2 derived from another spliceosomal protein HCF-1 that has not been detected in complexes containing CDC5L and PLRG1. The symbols on the right of the panel represent the input RNA, splicing intermediates and products. ( C ) Autoradiograph of a splicing gel from an experiment to determine the effect of overlapping 15mer peptides spanning the PL30-3 sequence on splicing. Similar amounts of peptide were added (lanes 4–12) to the splicing reactions as in (B). The lanes marked CTRL1 and CTRL2 contained splicing reactions treated in a similar way to lanes with the same names in (B). ( D ) Peptides containing the same amino acids as PL15-3 and PL30-3 in a scrambled sequence do not inhibit splicing. Lane 1, CTRL1 is the control reaction without peptide; lane 2, ∼20 nmol PL30-3; lanes 3–5, 7–20 nmol PL-SB15; lanes 6–8, 7–20 nmol PL-SB30 peptide. ( E ) Pull-down of CDC5L onto streptavidin–agarose beads from HeLa nuclear extract using PLRG1 peptides. Lanes 4–9, pull-down assays with the corresponding peptides (used in marking each lane). CTRL1 did not contain any peptides whereas CTRL2 contained a control peptide that does not inhibit splicing. The blot was probed with a buffer containing both anti-CDC5L and anti-PLRG1 antibodies. The arrows on the right of the panel show the positions of the CDC5L and PLRG1 proteins.

    Techniques Used: Binding Assay, Autoradiography, Derivative Assay, Sequencing

    23) Product Images from "Selective requirement of H2B N-Terminal tail for p14ARF-induced chromatin silencing"

    Article Title: Selective requirement of H2B N-Terminal tail for p14ARF-induced chromatin silencing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr642

    Mapping of the H2B and p14ARF interaction domains. ( A ) Preferential binding of p14ARF to H2B tail. Interaction of 14ARF with histone tails was examined by GST pull-down assays using GST (lane 2) or GST–histone tail fusions (lanes 3–6), and the binding reactions were analyzed by immunoblotting and Coomassie brilliant blue (CBB) staining. Lane 1 contains 50% of the input p14ARF. ( B ) p14ARF interaction with H2B tail deletion mutants. The left panel shows the schematic illustration of H2B tail and its deletion mutants. Numbers indicate amino acid residues. The right panel shows the detection of Flag-p14ARF in GST-pull down assays with GST (lane 2) or GST-H2B tail deletion mutants (lanes 3–7). ( C ) p14ARF interaction with H2B tail-deleted nucleosomes. Nucleosomes containing wild-type or tailless H2B were reconstituted on biotinylated 207 bp 601 fragments and immobilized on Streptavidin agarose beads. The binding assays were performed with Flag-p14ARF, and the presence of p14ARF in the beads was analyzed by immunoblotting with anti-Flag antibody. ( D ) Impairment of p14ARF-induced chromatin repression by H2B tail deletions. In vitro transcription experiments were performed as described in Figure 1 D, but with chromatin templates containing H2B N-terminal deletion mutants. ( E ) H2B tail interaction with p14ARF deletion mutants. The top panel shows the schematic illustration of p14ARF deletion mutants. The bottom panel shows the detection of p14ARF deletion mutants in GST pull-down assay with GST–H2B tail. Numbers indicate amino acid residues. Input lanes 1–3 represent 25% of p14ARF used in the binding reactions. ( F ) Nucleosome interaction with p14ARF deletion mutants. Nucleosomes containing wild-type H2B were reconstituted on biotinylated 207 bp 601 sequences and immobilized on Streptavidin agarose beads. The binding assays were performed with Flag-p14ARF deletion mutants, and the presence of p14ARF deletion mutants in the beads was analyzed by immunoblotting with anti-Flag antibody. ( G ) Effects of N- and C-terminal regions of p14ARF on chromatin transcription. Transcription reactions were essentially as described in Figure 1 D, but contained p14ARF deletion mutants. Heat-treated (HI) p14ARF proteins were also tested in control reactions.
    Figure Legend Snippet: Mapping of the H2B and p14ARF interaction domains. ( A ) Preferential binding of p14ARF to H2B tail. Interaction of 14ARF with histone tails was examined by GST pull-down assays using GST (lane 2) or GST–histone tail fusions (lanes 3–6), and the binding reactions were analyzed by immunoblotting and Coomassie brilliant blue (CBB) staining. Lane 1 contains 50% of the input p14ARF. ( B ) p14ARF interaction with H2B tail deletion mutants. The left panel shows the schematic illustration of H2B tail and its deletion mutants. Numbers indicate amino acid residues. The right panel shows the detection of Flag-p14ARF in GST-pull down assays with GST (lane 2) or GST-H2B tail deletion mutants (lanes 3–7). ( C ) p14ARF interaction with H2B tail-deleted nucleosomes. Nucleosomes containing wild-type or tailless H2B were reconstituted on biotinylated 207 bp 601 fragments and immobilized on Streptavidin agarose beads. The binding assays were performed with Flag-p14ARF, and the presence of p14ARF in the beads was analyzed by immunoblotting with anti-Flag antibody. ( D ) Impairment of p14ARF-induced chromatin repression by H2B tail deletions. In vitro transcription experiments were performed as described in Figure 1 D, but with chromatin templates containing H2B N-terminal deletion mutants. ( E ) H2B tail interaction with p14ARF deletion mutants. The top panel shows the schematic illustration of p14ARF deletion mutants. The bottom panel shows the detection of p14ARF deletion mutants in GST pull-down assay with GST–H2B tail. Numbers indicate amino acid residues. Input lanes 1–3 represent 25% of p14ARF used in the binding reactions. ( F ) Nucleosome interaction with p14ARF deletion mutants. Nucleosomes containing wild-type H2B were reconstituted on biotinylated 207 bp 601 sequences and immobilized on Streptavidin agarose beads. The binding assays were performed with Flag-p14ARF deletion mutants, and the presence of p14ARF deletion mutants in the beads was analyzed by immunoblotting with anti-Flag antibody. ( G ) Effects of N- and C-terminal regions of p14ARF on chromatin transcription. Transcription reactions were essentially as described in Figure 1 D, but contained p14ARF deletion mutants. Heat-treated (HI) p14ARF proteins were also tested in control reactions.

    Techniques Used: Binding Assay, Staining, In Vitro, Pull Down Assay

    Regulation of p14ARF function by H2B–K20 acetylation. ( A ) Antagonistic effects of H2B–K20 acetylation on p14ARF transcriptional repression. Transcription assays were performed as in Figure 1 D, but p300 was added to the reactions before p14ARF. Chromatin templates contain wild-type (WT) (lanes 1–5) or lysine-mutated (K5R, K12, 15R, K20R and K5, 12, 15, 20R) (lanes 6–25) H2B proteins. ( B ) Antagonistic effects of K20 acetylation on H2B tail–p14ARF interaction. Flag-tagged p14ARF was tested for binding to GST (lanes 2 and 3) or GST-fused wild-type (WT) or lysine-mutated (K5R, K12,15R, K20R and K5,12,15,20R) H2B tails (lanes 4–9). Lane 1 shows 25% of p14ARF used in the binding reactions. Experiments were repeated three times with comparable results. Data were quantitated by phosphoimager analysis. ( C ) Antagonistic effects of H2B–K20 acetylation on nucleosome-p14ARF interaction. Mononucleosomes containing wild-type (WT) or mutant (K20R and K5,12,15,20R) H2B were reconstituted on biotinylated 207 bp G5ML fragments and immobilized on Streptavidin agarose beads. Flag-tagged p14ARF was incubated with the nucleosomes containing wild-type (lanes 1 and 2) or mutant (lanes 3–6) H2B, and p14ARF binding was determined by immunoblot analysis using anti-Flag antibody. Data were quantitated by phosphoimager analysis, and similar results were obtained in two independent binding experiments.
    Figure Legend Snippet: Regulation of p14ARF function by H2B–K20 acetylation. ( A ) Antagonistic effects of H2B–K20 acetylation on p14ARF transcriptional repression. Transcription assays were performed as in Figure 1 D, but p300 was added to the reactions before p14ARF. Chromatin templates contain wild-type (WT) (lanes 1–5) or lysine-mutated (K5R, K12, 15R, K20R and K5, 12, 15, 20R) (lanes 6–25) H2B proteins. ( B ) Antagonistic effects of K20 acetylation on H2B tail–p14ARF interaction. Flag-tagged p14ARF was tested for binding to GST (lanes 2 and 3) or GST-fused wild-type (WT) or lysine-mutated (K5R, K12,15R, K20R and K5,12,15,20R) H2B tails (lanes 4–9). Lane 1 shows 25% of p14ARF used in the binding reactions. Experiments were repeated three times with comparable results. Data were quantitated by phosphoimager analysis. ( C ) Antagonistic effects of H2B–K20 acetylation on nucleosome-p14ARF interaction. Mononucleosomes containing wild-type (WT) or mutant (K20R and K5,12,15,20R) H2B were reconstituted on biotinylated 207 bp G5ML fragments and immobilized on Streptavidin agarose beads. Flag-tagged p14ARF was incubated with the nucleosomes containing wild-type (lanes 1 and 2) or mutant (lanes 3–6) H2B, and p14ARF binding was determined by immunoblot analysis using anti-Flag antibody. Data were quantitated by phosphoimager analysis, and similar results were obtained in two independent binding experiments.

    Techniques Used: Binding Assay, Mutagenesis, Incubation

    24) Product Images from "α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells"

    Article Title: α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2009.84

    c-Met is hyposialylated in ST6Gal-I–null HCT116 cells. (A) ST6Gal-I knockdown in HCT116 cells caused c-Met hyposialylation. Equivalent amounts of cell lysates were incubated with SNA. SNA-glycoprotein complexes were precipitated with streptavidin-conjugated agarose beads and probed by immunoblotting with specific antibody. (B) ST6Gal-I-KD HCT116 cells failed to migrate in response to HGF stimulation. The migration ability of D3, NC, and P cells to HGF stimulation was assessed using a Boyden chamber assay. (C) The migrated cells on the lower surface of the membrane were captured, and five random fields were analyzed using Image-Pro Plus image analysis software. c P
    Figure Legend Snippet: c-Met is hyposialylated in ST6Gal-I–null HCT116 cells. (A) ST6Gal-I knockdown in HCT116 cells caused c-Met hyposialylation. Equivalent amounts of cell lysates were incubated with SNA. SNA-glycoprotein complexes were precipitated with streptavidin-conjugated agarose beads and probed by immunoblotting with specific antibody. (B) ST6Gal-I-KD HCT116 cells failed to migrate in response to HGF stimulation. The migration ability of D3, NC, and P cells to HGF stimulation was assessed using a Boyden chamber assay. (C) The migrated cells on the lower surface of the membrane were captured, and five random fields were analyzed using Image-Pro Plus image analysis software. c P

    Techniques Used: Incubation, Migration, Boyden Chamber Assay, Software

    D3 siRNA transfection does not affect cell surface expression of α2,3-sialic acid structures. Adherent D3, NC, and P cells were first released by trypsinization and then labeled with 2 μg MAA-biotin. After incubation with 0.25 μg RPE-conjugated streptavidin, labeled cells were subjected to FACS analysis. The data are representative of three independent experiments with similar results.
    Figure Legend Snippet: D3 siRNA transfection does not affect cell surface expression of α2,3-sialic acid structures. Adherent D3, NC, and P cells were first released by trypsinization and then labeled with 2 μg MAA-biotin. After incubation with 0.25 μg RPE-conjugated streptavidin, labeled cells were subjected to FACS analysis. The data are representative of three independent experiments with similar results.

    Techniques Used: Transfection, Expressing, Labeling, Incubation, FACS

    25) Product Images from "Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein"

    Article Title: Comprehensive Mutational Analysis of the Moloney Murine Leukemia Virus Envelope Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.23.11851-11862.2001

    Overview of genetic footprinting. (A) Selection and analysis. A pool of single insertion variants of a gene is subjected to a selection for gene function. The resulting pre- and postselection pools are analyzed by a PCR approach that generates a uniquely sized fragment for each mutant, reflecting the position of the insertion in the gene sequence. Polyacrylamide gel electrophoresis of the fragments generates a footprint, corresponding to an essential region of the gene in which insertions decrease function. Symbols: B→, biotinylated primer; ←✽, radiolabeled primer; SA, streptavidin-agarose. (B) Sequence of the insertion site. Duplication of the target sequence (XXXXX) results in the net in-frame insertion of 15 nucleotides. The identity of the inserted amino acids depends on the relative frame of the insert within the target gene sequence.
    Figure Legend Snippet: Overview of genetic footprinting. (A) Selection and analysis. A pool of single insertion variants of a gene is subjected to a selection for gene function. The resulting pre- and postselection pools are analyzed by a PCR approach that generates a uniquely sized fragment for each mutant, reflecting the position of the insertion in the gene sequence. Polyacrylamide gel electrophoresis of the fragments generates a footprint, corresponding to an essential region of the gene in which insertions decrease function. Symbols: B→, biotinylated primer; ←✽, radiolabeled primer; SA, streptavidin-agarose. (B) Sequence of the insertion site. Duplication of the target sequence (XXXXX) results in the net in-frame insertion of 15 nucleotides. The identity of the inserted amino acids depends on the relative frame of the insert within the target gene sequence.

    Techniques Used: Footprinting, Selection, Polymerase Chain Reaction, Mutagenesis, Sequencing, Polyacrylamide Gel Electrophoresis

    26) Product Images from "Functional characterization of wild-type and mutant human sialin"

    Article Title: Functional characterization of wild-type and mutant human sialin

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600464

    Mutation of the DRTPLL motif results in cell surface expression. HEK293 cells transiently expressing GFP (lanes 1–3), WT GFP-sialin (lanes 4–6) or GFP-sialin L22G/L23G (lanes 7–9) were treated with an impermeant biotinylation reagent. After cell lysis, biotinylated proteins were purified on streptavidin–agarose beads. In lanes 10–12, biotinylation was omitted to verify the selectivity of the affinity chromatography. Equal amounts of cell lysate (tot) and streptavidin-unbound material (ub) were analysed by SDS–PAGE and immunoblotting using anti-GFP antibodies. Streptavidin-bound proteins (bo) were derived from an 18-fold higher amount of material. The position and molecular mass (kDa) of protein standards is shown on the left.
    Figure Legend Snippet: Mutation of the DRTPLL motif results in cell surface expression. HEK293 cells transiently expressing GFP (lanes 1–3), WT GFP-sialin (lanes 4–6) or GFP-sialin L22G/L23G (lanes 7–9) were treated with an impermeant biotinylation reagent. After cell lysis, biotinylated proteins were purified on streptavidin–agarose beads. In lanes 10–12, biotinylation was omitted to verify the selectivity of the affinity chromatography. Equal amounts of cell lysate (tot) and streptavidin-unbound material (ub) were analysed by SDS–PAGE and immunoblotting using anti-GFP antibodies. Streptavidin-bound proteins (bo) were derived from an 18-fold higher amount of material. The position and molecular mass (kDa) of protein standards is shown on the left.

    Techniques Used: Mutagenesis, Expressing, Lysis, Purification, Affinity Chromatography, SDS Page, Derivative Assay

    27) Product Images from "Identification of Piperazinylbenzenesulfonamides as New Inhibitors of Claudin-1 Trafficking and Hepatitis C Virus Entry"

    Article Title: Identification of Piperazinylbenzenesulfonamides as New Inhibitors of Claudin-1 Trafficking and Hepatitis C Virus Entry

    Journal: Journal of Virology

    doi: 10.1128/JVI.01982-17

    SB258585 alters CLDN1 recycling, causing its intracellular accumulation. (A) Huh-7 cells were treated for 2 h with DMSO or increasing concentrations of SB258585. Cell surface expression of CD81 and CLDN1 was analyzed by immunofluorescence assay. Images were taken using a Zeiss LSM-880 microscope and a 63× objective. (B) Pearson correlation coefficients (PCCs) were calculated for cell surface ROIs for at least 40 different cells for each condition. (C) Huh-7 cells were treated for 2 h with DMSO or increasing concentrations of SB258585, and CLDN1 expression was analyzed by flow cytometry. Curves from a representative experiment are shown. Mean fluorescence intensities (MFI) relative to that for the DMSO-treated condition are also presented. (D) Huh-7 cells were treated for 2 h with DMSO or increasing concentrations of SB399885, and CLDN1 expression was analyzed by flow cytometry. (E) Huh-7 cells were incubated with SB258585 (100 μM) for the indicated periods. CLDN1 present at the cell surface was quantified by flow cytometry. (F) Huh-7 cells were treated for 2 h with SB258585 (100 μM). The drug was then removed and replaced by DMEM for the indicated times. Cytometry analyses were performed to quantify CLDN1 at the cell surface. For panels D to F, mean fluorescence intensities relative to those for the DMSO-treated condition are shown. (G) Huh-7 cells were treated for 2 h with DMSO, SB258585 (100 μM), SB399885 (100 μM), or H89 (10 μM). The total quantity of CLDN1 was assessed by Western blotting. β-Tubulin was used as a loading control. (H) Huh-7 cells were treated for 2 h with DMSO, SB258585 (100 μM), or H89 (10 μM). CLDN1 subcellular localization was determined by immunofluorescence assay after membrane permeabilization. Images were taken with a 63× objective. (I) TGN46 was stained concomitantly with CLDN1, and PCCs were calculated for intracellular CLDN1-TGN46 colocalization for > 35 cells for each condition. (J) After surface biotinylation, Huh-7 cells were incubated at 37°C with DMSO or SB258585 (100 μM) for the indicated times. Biotin remaining at the cell surface was cleaved by use of glutathione. The amount of internalized CLDN1 was determined by Western blotting after pulldown of biotin-labeled proteins with streptavidin-agarose beads. A representative Western blot ( n = 3) is presented. All results are presented as means ± SEM ( n = 3). One-way ANOVA (B to E and I) or two-way ANOVA (F) followed by the Dunnett or Bonferroni posttest was performed for statistical analysis. *, P
    Figure Legend Snippet: SB258585 alters CLDN1 recycling, causing its intracellular accumulation. (A) Huh-7 cells were treated for 2 h with DMSO or increasing concentrations of SB258585. Cell surface expression of CD81 and CLDN1 was analyzed by immunofluorescence assay. Images were taken using a Zeiss LSM-880 microscope and a 63× objective. (B) Pearson correlation coefficients (PCCs) were calculated for cell surface ROIs for at least 40 different cells for each condition. (C) Huh-7 cells were treated for 2 h with DMSO or increasing concentrations of SB258585, and CLDN1 expression was analyzed by flow cytometry. Curves from a representative experiment are shown. Mean fluorescence intensities (MFI) relative to that for the DMSO-treated condition are also presented. (D) Huh-7 cells were treated for 2 h with DMSO or increasing concentrations of SB399885, and CLDN1 expression was analyzed by flow cytometry. (E) Huh-7 cells were incubated with SB258585 (100 μM) for the indicated periods. CLDN1 present at the cell surface was quantified by flow cytometry. (F) Huh-7 cells were treated for 2 h with SB258585 (100 μM). The drug was then removed and replaced by DMEM for the indicated times. Cytometry analyses were performed to quantify CLDN1 at the cell surface. For panels D to F, mean fluorescence intensities relative to those for the DMSO-treated condition are shown. (G) Huh-7 cells were treated for 2 h with DMSO, SB258585 (100 μM), SB399885 (100 μM), or H89 (10 μM). The total quantity of CLDN1 was assessed by Western blotting. β-Tubulin was used as a loading control. (H) Huh-7 cells were treated for 2 h with DMSO, SB258585 (100 μM), or H89 (10 μM). CLDN1 subcellular localization was determined by immunofluorescence assay after membrane permeabilization. Images were taken with a 63× objective. (I) TGN46 was stained concomitantly with CLDN1, and PCCs were calculated for intracellular CLDN1-TGN46 colocalization for > 35 cells for each condition. (J) After surface biotinylation, Huh-7 cells were incubated at 37°C with DMSO or SB258585 (100 μM) for the indicated times. Biotin remaining at the cell surface was cleaved by use of glutathione. The amount of internalized CLDN1 was determined by Western blotting after pulldown of biotin-labeled proteins with streptavidin-agarose beads. A representative Western blot ( n = 3) is presented. All results are presented as means ± SEM ( n = 3). One-way ANOVA (B to E and I) or two-way ANOVA (F) followed by the Dunnett or Bonferroni posttest was performed for statistical analysis. *, P

    Techniques Used: Expressing, Immunofluorescence, Microscopy, Flow Cytometry, Cytometry, Fluorescence, Incubation, Western Blot, Staining, Labeling

    28) Product Images from "Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress"

    Article Title: Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress

    Journal: Diseases

    doi: 10.3390/diseases6030064

    Analysis of viral PPxY-host WW-domain interactions between BAG3 and LFV-Z by peptide pull-down assays. ( A ) Flow chart of the peptide pull-down assay using LFV-Z peptides and cell lysates expressing BAG3-WT; ( B ) Schematic diagram of BAG3-WT, BAG3-ΔN, and BAG3-ΔC mutants with the various domains highlighted in color and amino acid positions indicated; ( C ) Western blot of peptide pull-down assay using streptavidin agarose beads conjugated with either the LFV-Z WT or LFV-Z PY mutant peptide. BAG3 proteins were detected using anti-c-myc antibody (top blot). Expression controls for BAG3 and actin are shown in the bottom blot. These results are from 1 of 2 independent experiments.
    Figure Legend Snippet: Analysis of viral PPxY-host WW-domain interactions between BAG3 and LFV-Z by peptide pull-down assays. ( A ) Flow chart of the peptide pull-down assay using LFV-Z peptides and cell lysates expressing BAG3-WT; ( B ) Schematic diagram of BAG3-WT, BAG3-ΔN, and BAG3-ΔC mutants with the various domains highlighted in color and amino acid positions indicated; ( C ) Western blot of peptide pull-down assay using streptavidin agarose beads conjugated with either the LFV-Z WT or LFV-Z PY mutant peptide. BAG3 proteins were detected using anti-c-myc antibody (top blot). Expression controls for BAG3 and actin are shown in the bottom blot. These results are from 1 of 2 independent experiments.

    Techniques Used: Flow Cytometry, Pull Down Assay, Expressing, Western Blot, Mutagenesis

    29) Product Images from "Caspase-9 mediates synaptic plasticity and memory deficits of Danish dementia knock-in mice: caspase-9 inhibition provides therapeutic protection"

    Article Title: Caspase-9 mediates synaptic plasticity and memory deficits of Danish dementia knock-in mice: caspase-9 inhibition provides therapeutic protection

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-7-60

    High levels of active initiator caspase-9 in FDD KI mice. A , Homogenates (input) were prepared from the bVAD injected (+bVAD) and contralateral non-injected (con.) hippocampal regions of WT and FDD KI mice. Active caspases were isolated from homogenates with streptavidin-agarose-beads pull-down. Western blot analysis shows that the caspase inhibitor bVAD traps FL-caspase-9 only from the bVAD injected FDD KI mouse hippocampus; FL-caspase-8, cl.caspase-3 and cl.-caspase-6 are not trapped. B , In a similar experiment, the streptavidin-agarose-beads pull-down experiment was performed from the P2 fractions. The P2 fractions represent crude synaptosomal fractions (see Material and Methods for details about these preparations). Again, active FL-caspase-9 is isolated from FDD KI but not WT mice. C , Organotypic hippocampal cultures from either FDD KI or WT mice were incubated for 3 hrs with 45 μM bVAD. After lysis, active caspases were isolated from homogenates. Again, caspase-9 was the only active caspase isolated. Albeit traces of active caspase-9 are found in the WT samples, the levels found in the FDD KI hippocampus are greatly elevated. The blots shown in A, B and C are representative of duplicate experiments.
    Figure Legend Snippet: High levels of active initiator caspase-9 in FDD KI mice. A , Homogenates (input) were prepared from the bVAD injected (+bVAD) and contralateral non-injected (con.) hippocampal regions of WT and FDD KI mice. Active caspases were isolated from homogenates with streptavidin-agarose-beads pull-down. Western blot analysis shows that the caspase inhibitor bVAD traps FL-caspase-9 only from the bVAD injected FDD KI mouse hippocampus; FL-caspase-8, cl.caspase-3 and cl.-caspase-6 are not trapped. B , In a similar experiment, the streptavidin-agarose-beads pull-down experiment was performed from the P2 fractions. The P2 fractions represent crude synaptosomal fractions (see Material and Methods for details about these preparations). Again, active FL-caspase-9 is isolated from FDD KI but not WT mice. C , Organotypic hippocampal cultures from either FDD KI or WT mice were incubated for 3 hrs with 45 μM bVAD. After lysis, active caspases were isolated from homogenates. Again, caspase-9 was the only active caspase isolated. Albeit traces of active caspase-9 are found in the WT samples, the levels found in the FDD KI hippocampus are greatly elevated. The blots shown in A, B and C are representative of duplicate experiments.

    Techniques Used: Mouse Assay, Injection, Isolation, Western Blot, Incubation, Lysis

    30) Product Images from "Assembly with the NR1 Subunit Is Required for Surface Expression of NR3A-Containing NMDA Receptors"

    Article Title: Assembly with the NR1 Subunit Is Required for Surface Expression of NR3A-Containing NMDA Receptors

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-04-01228.2001

    NR3A is present at the cell surface only when coexpressed with the NR1-1a subunit. A , HEK293T cells were transfected with different combinations of NMDA receptor subunits and incubated for 15 min with sulfo-NHS-biotin. After solubilization, biotinylated protein was recovered by streptavidin precipitation. The streptavidin fractions ( lanes labeled 2 ), representing the membrane proteins, and aliquots of the lysate before ( lanes labeled 1 ) and after ( lanes labeled 3 ) streptavidin precipitation were analyzed by immunoblotting using anti-NR1, anti-NR2A/B, anti-NR3A, and anti-calreticulin antibodies. An excess amount of protein was loaded in the lanes labeled 2 to ensure detection of any NR3A or calreticulin at the cell surface. The subunit combinations used for transfection are indicated above each blot, and the positions of molecular size markers in kilodaltons are shown on the left . A representative experiment is shown; n = 3. B, C , Surface localization of GFP-tagged NR3A. B, Left , Schematic drawing of expected transmembrane ( TM ) topology of NR3A-GFP is shown. Right , Protein immunoblots of HEK293T cells transfected with NR3A or NR3AGFP and probed with anti-NR3A antibody show an increase in NR3A molecular weight that corresponds to the molecular mass of GFP (27 kDa). No lower molecular weight bands were observed. C , Cells transfected with GFP-tagged NR3A alone or in combination with the other NMDA receptor subunits were immunostained in nonpermeabilizing (NP) conditions with anti-GFP antibody followed by a Texas Red-conjugated secondary antibody and imaged with filters for GFP and Texas Red. All four panels show raw superimposed confocal images combining NP anti-GFP antibody staining ( red ) and native GFP fluorescence from NR3A-GFP ( green ). Yellow corresponds to the overlap of GFP immunostaining and GFP fluorescence and reflects NR3A-GFP expressed at the cell surface. Because the intensity of red immunostaining was brighter than was green GFP fluorescence, regions of overlapping can appear red-yellow . When expressed alone, NR3A-GFP exhibits a perinuclear and reticular fluorescence pattern, and no surface staining is observed. Cotransfection of NR1-1a/NR2A leads to the appearance of patches of fluorescence at the plasma membrane. Scale bar, 10 μm.
    Figure Legend Snippet: NR3A is present at the cell surface only when coexpressed with the NR1-1a subunit. A , HEK293T cells were transfected with different combinations of NMDA receptor subunits and incubated for 15 min with sulfo-NHS-biotin. After solubilization, biotinylated protein was recovered by streptavidin precipitation. The streptavidin fractions ( lanes labeled 2 ), representing the membrane proteins, and aliquots of the lysate before ( lanes labeled 1 ) and after ( lanes labeled 3 ) streptavidin precipitation were analyzed by immunoblotting using anti-NR1, anti-NR2A/B, anti-NR3A, and anti-calreticulin antibodies. An excess amount of protein was loaded in the lanes labeled 2 to ensure detection of any NR3A or calreticulin at the cell surface. The subunit combinations used for transfection are indicated above each blot, and the positions of molecular size markers in kilodaltons are shown on the left . A representative experiment is shown; n = 3. B, C , Surface localization of GFP-tagged NR3A. B, Left , Schematic drawing of expected transmembrane ( TM ) topology of NR3A-GFP is shown. Right , Protein immunoblots of HEK293T cells transfected with NR3A or NR3AGFP and probed with anti-NR3A antibody show an increase in NR3A molecular weight that corresponds to the molecular mass of GFP (27 kDa). No lower molecular weight bands were observed. C , Cells transfected with GFP-tagged NR3A alone or in combination with the other NMDA receptor subunits were immunostained in nonpermeabilizing (NP) conditions with anti-GFP antibody followed by a Texas Red-conjugated secondary antibody and imaged with filters for GFP and Texas Red. All four panels show raw superimposed confocal images combining NP anti-GFP antibody staining ( red ) and native GFP fluorescence from NR3A-GFP ( green ). Yellow corresponds to the overlap of GFP immunostaining and GFP fluorescence and reflects NR3A-GFP expressed at the cell surface. Because the intensity of red immunostaining was brighter than was green GFP fluorescence, regions of overlapping can appear red-yellow . When expressed alone, NR3A-GFP exhibits a perinuclear and reticular fluorescence pattern, and no surface staining is observed. Cotransfection of NR1-1a/NR2A leads to the appearance of patches of fluorescence at the plasma membrane. Scale bar, 10 μm.

    Techniques Used: Transfection, Incubation, Labeling, Western Blot, Molecular Weight, Staining, Fluorescence, Immunostaining, Cotransfection

    31) Product Images from "Sequestration by IFIT1 Impairs Translation of 2?O-unmethylated Capped RNA"

    Article Title: Sequestration by IFIT1 Impairs Translation of 2?O-unmethylated Capped RNA

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003663

    Mass spectrometry-based identification of human and murine interactors of capped RNA. ( a ) Schematic depiction of the experimental approach used for mass spectrometry (MS)-based identification of cellular RNA binding proteins. Biotinylated RNA with different 5′ end structures (OH, PPP, CAP, CAP0, CAP1) was coupled to streptavidin beads, and incubated with lysates obtained from cells that had been left untreated or treated with 1000 U/ml IFN-α for 16 h. Bound proteins were denatured, alkylated and directly digested with trypsin. The resulting peptides were subjected to shotgun liquid chromatography-tandem MS (LC-MS/MS). Three independent experiments were performed for each RNA bait, and the data were analysed with the MaxQuant software [37] using the label-free quantification algorithm [38] . ( b–d ) Proteins obtained from lysates of IFN-α-treated HeLa cells using the indicated biotinylated RNA baits were analysed by LC-MS/MS. Volcano plots show the degrees of enrichment (ratio of label-free quantitation (LFQ) protein intensities; x-axis) and p-values (t-test; y-axis) by PPP-RNA ( b ), CAP-RNA ( c ), and CAP1-RNA ( d ) baits as compared to OH-RNA. Significantly enriched interactors (see Materials and Methods ) are separated by a hyperbolic curve (dotted line) from background proteins (blue dots), known cap-binding proteins (dark-green), and proteins known to associate with capped RNA (light green). Interferon-induced proteins [21] detected in the significantly enriched fractions (IFIT1-3 and 5, DDX58) are highlighted (red triangles). ( e–g ) As in ( b–d ) but for lysates of IFN-α-treated mouse embryo fibroblasts (MEFs). The interferon-induced proteins Ifit1 and Ifit1c [42] in significantly enriched and non-enriched fractions are highlighted.
    Figure Legend Snippet: Mass spectrometry-based identification of human and murine interactors of capped RNA. ( a ) Schematic depiction of the experimental approach used for mass spectrometry (MS)-based identification of cellular RNA binding proteins. Biotinylated RNA with different 5′ end structures (OH, PPP, CAP, CAP0, CAP1) was coupled to streptavidin beads, and incubated with lysates obtained from cells that had been left untreated or treated with 1000 U/ml IFN-α for 16 h. Bound proteins were denatured, alkylated and directly digested with trypsin. The resulting peptides were subjected to shotgun liquid chromatography-tandem MS (LC-MS/MS). Three independent experiments were performed for each RNA bait, and the data were analysed with the MaxQuant software [37] using the label-free quantification algorithm [38] . ( b–d ) Proteins obtained from lysates of IFN-α-treated HeLa cells using the indicated biotinylated RNA baits were analysed by LC-MS/MS. Volcano plots show the degrees of enrichment (ratio of label-free quantitation (LFQ) protein intensities; x-axis) and p-values (t-test; y-axis) by PPP-RNA ( b ), CAP-RNA ( c ), and CAP1-RNA ( d ) baits as compared to OH-RNA. Significantly enriched interactors (see Materials and Methods ) are separated by a hyperbolic curve (dotted line) from background proteins (blue dots), known cap-binding proteins (dark-green), and proteins known to associate with capped RNA (light green). Interferon-induced proteins [21] detected in the significantly enriched fractions (IFIT1-3 and 5, DDX58) are highlighted (red triangles). ( e–g ) As in ( b–d ) but for lysates of IFN-α-treated mouse embryo fibroblasts (MEFs). The interferon-induced proteins Ifit1 and Ifit1c [42] in significantly enriched and non-enriched fractions are highlighted.

    Techniques Used: Mass Spectrometry, RNA Binding Assay, Incubation, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Software, Quantitation Assay, T-Test, Binding Assay

    Competition between IFIT1 and translation factor EIF4E for mRNA templates. ( a ) Recovery of recombinant human EIF4E based on RNA affinity binding in the presence or absence of IFIT1. Streptavidin beads were coupled to 250 ng of the indicated RNA and mixed with 5 µg of recombinant His-tagged hIFIT1 and/or His-tagged EIF4E, as indicated. Bound proteins were analysed by western blotting with antibodies directed against the His-tag. ( b ) As in ( a ), except that RNA-coated beads were incubated with lysates of interferon-treated Ifit1 +/+ and Ifit1 −/− mouse embryo fibroblasts. Bound proteins were analysed by western blotting with antibodies directed against murine Eif4e and mIfit1. ( c ) Proposed model for IFIT1-mediated translational inhibition of 2′O-unmethylated viral RNA. Capped and 2′O-methylated cellular and viral RNA is bound by EIF4E to initiate translation. Viral mRNA lacking 2′O methylation at the first ribose is recognized by IFIT1 which prevents binding of cellular factors required for efficient translation. The model is based on data presented here and elsewhere [16] , [17] , [19] , [20] .
    Figure Legend Snippet: Competition between IFIT1 and translation factor EIF4E for mRNA templates. ( a ) Recovery of recombinant human EIF4E based on RNA affinity binding in the presence or absence of IFIT1. Streptavidin beads were coupled to 250 ng of the indicated RNA and mixed with 5 µg of recombinant His-tagged hIFIT1 and/or His-tagged EIF4E, as indicated. Bound proteins were analysed by western blotting with antibodies directed against the His-tag. ( b ) As in ( a ), except that RNA-coated beads were incubated with lysates of interferon-treated Ifit1 +/+ and Ifit1 −/− mouse embryo fibroblasts. Bound proteins were analysed by western blotting with antibodies directed against murine Eif4e and mIfit1. ( c ) Proposed model for IFIT1-mediated translational inhibition of 2′O-unmethylated viral RNA. Capped and 2′O-methylated cellular and viral RNA is bound by EIF4E to initiate translation. Viral mRNA lacking 2′O methylation at the first ribose is recognized by IFIT1 which prevents binding of cellular factors required for efficient translation. The model is based on data presented here and elsewhere [16] , [17] , [19] , [20] .

    Techniques Used: Recombinant, Binding Assay, Western Blot, Incubation, Inhibition, Methylation

    32) Product Images from "ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation"

    Article Title: ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation

    Journal: Nature Communications

    doi: 10.1038/ncomms11733

    Impact of ZBTB7A mutations on DNA binding. ( a ) Model for the C-terminal zinc-finger domain of ZBTB7A comprising residues 382–488. The model is depicted as yellow ribbon with highlighted secondary structure. Zinc ions are shown as grey spheres. DNA is shown in brown with a grey molecular surface. R402 (purple) binds into the major groove and likely contributes to the affinity or sequence specificity of the DNA interaction of the zinc-finger domain. ( b ) Biotinylated oligonucleotides containing the ZBTB7A (alias: Pokemon) consensus binding motif (POK WT) or a mutant thereof (POK mut) 14 used in DNA pull-down experiments. Spheres illustrate streptavidin-coated beads. ( c ) DNA pull-down using protein lysates from HEK293T cells expressing wild-type or mutant ZBTB7A. Western blot analysis shows that A175fs and R402H fail to bind oligonutides with a ZBTB7A-binding site (POK WT). Oligonucleotides with a mutated binding site (POK mut) were used as negative control. Input lanes were loaded with 10% of the protein lysate used for each binding reaction.
    Figure Legend Snippet: Impact of ZBTB7A mutations on DNA binding. ( a ) Model for the C-terminal zinc-finger domain of ZBTB7A comprising residues 382–488. The model is depicted as yellow ribbon with highlighted secondary structure. Zinc ions are shown as grey spheres. DNA is shown in brown with a grey molecular surface. R402 (purple) binds into the major groove and likely contributes to the affinity or sequence specificity of the DNA interaction of the zinc-finger domain. ( b ) Biotinylated oligonucleotides containing the ZBTB7A (alias: Pokemon) consensus binding motif (POK WT) or a mutant thereof (POK mut) 14 used in DNA pull-down experiments. Spheres illustrate streptavidin-coated beads. ( c ) DNA pull-down using protein lysates from HEK293T cells expressing wild-type or mutant ZBTB7A. Western blot analysis shows that A175fs and R402H fail to bind oligonutides with a ZBTB7A-binding site (POK WT). Oligonucleotides with a mutated binding site (POK mut) were used as negative control. Input lanes were loaded with 10% of the protein lysate used for each binding reaction.

    Techniques Used: Binding Assay, Sequencing, Mutagenesis, Expressing, Western Blot, Negative Control

    33) Product Images from "TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis"

    Article Title: TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-018-0221-7

    TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P
    Figure Legend Snippet: TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P

    Techniques Used: Expressing, Sequencing, Binding Assay, Fractionation, Luciferase, Activity Assay, Transfection, Labeling, Pull Down Assay, Quantitative RT-PCR

    34) Product Images from "HAP1 Is Required for Endocytosis and Signalling of BDNF and Its Receptors in Neurons"

    Article Title: HAP1 Is Required for Endocytosis and Signalling of BDNF and Its Receptors in Neurons

    Journal: Molecular neurobiology

    doi: 10.1007/s12035-016-0379-0

    Endocytosis of TrkB and p75 NTR in cortical neurons upon BDNF stimulation. a Western blots showing BDNF-induced endocytosis of TrkB and p75 NTR . HAP1 WT and KO cortical neurons, after 72-h culture, were stimulated with 0 (control) or 100 ng/ml BDNF for 30 min. Surface TrkB-FL, TrkB-T1 and p75 NTR were detected by Western blotting using anti-TrkB and anti-p75 NTR antibodies, respectively, after surface protein biotinylation and fractionation of biotinylated proteins with streptavidin-agarose. Total TrkB-FL, TrkB-T1, p75 NTR and β-actin were determined using whole cell lysates by Western blot with anti-TrkB, anti-p75 NTR and anti-β-actin antibody, respectively. b , c , d Each dot or square represents individual animal. Densitometric ratios of surface/total TrkB-FL levels ( b ), surface/total TrkB-T1 levels ( c ) and surface/total p75 NTR levels ( c ) assessed by biotinylation assay. Data presented as mean ± SEM ( n = 3 per group, Student’s t test). ‘WT control was normalised to 1. TrkB-FL, full-length of TrkB; TrkB-T1, truncated TrkB
    Figure Legend Snippet: Endocytosis of TrkB and p75 NTR in cortical neurons upon BDNF stimulation. a Western blots showing BDNF-induced endocytosis of TrkB and p75 NTR . HAP1 WT and KO cortical neurons, after 72-h culture, were stimulated with 0 (control) or 100 ng/ml BDNF for 30 min. Surface TrkB-FL, TrkB-T1 and p75 NTR were detected by Western blotting using anti-TrkB and anti-p75 NTR antibodies, respectively, after surface protein biotinylation and fractionation of biotinylated proteins with streptavidin-agarose. Total TrkB-FL, TrkB-T1, p75 NTR and β-actin were determined using whole cell lysates by Western blot with anti-TrkB, anti-p75 NTR and anti-β-actin antibody, respectively. b , c , d Each dot or square represents individual animal. Densitometric ratios of surface/total TrkB-FL levels ( b ), surface/total TrkB-T1 levels ( c ) and surface/total p75 NTR levels ( c ) assessed by biotinylation assay. Data presented as mean ± SEM ( n = 3 per group, Student’s t test). ‘WT control was normalised to 1. TrkB-FL, full-length of TrkB; TrkB-T1, truncated TrkB

    Techniques Used: Western Blot, Fractionation, Cell Surface Biotinylation Assay

    35) Product Images from "The tuberous sclerosis complex subunit TBC1D7 is stabilized by Akt phosphorylation–mediated 14-3-3 binding"

    Article Title: The tuberous sclerosis complex subunit TBC1D7 is stabilized by Akt phosphorylation–mediated 14-3-3 binding

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003525

    PHLPP proteins and TBC1D7 are binding partners. A , 293T cells were transfected with either SBP or SBP-PHLPP1 expression plasmids. Lysates were subject to pulldown analysis using streptavidin beads. Affinity-purified complexes were resolved on SDS-PAGE,
    Figure Legend Snippet: PHLPP proteins and TBC1D7 are binding partners. A , 293T cells were transfected with either SBP or SBP-PHLPP1 expression plasmids. Lysates were subject to pulldown analysis using streptavidin beads. Affinity-purified complexes were resolved on SDS-PAGE,

    Techniques Used: Binding Assay, Transfection, Expressing, Affinity Purification, SDS Page

    Ser-124 phosphorylation stabilizes TBC1D7. A , 293T cells were transfected with either SBP vector or SBP-TBC1D7 WT, S124A, S124E expression plasmids. Lysates were subject to pulldown analysis with streptavidin beads. Affinity-purified complexes and input
    Figure Legend Snippet: Ser-124 phosphorylation stabilizes TBC1D7. A , 293T cells were transfected with either SBP vector or SBP-TBC1D7 WT, S124A, S124E expression plasmids. Lysates were subject to pulldown analysis with streptavidin beads. Affinity-purified complexes and input

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Affinity Purification

    36) Product Images from "Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway"

    Article Title: Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115701

    Stimulation of α 1A -AR transduced HEK-293/EBNA with A-61603 and Iso leads to an increase in intracellular α 1A – AR. A . HEK293 cells were transiently transfected with α 1A – AR only (top panels), or co-transfected with α 1A – AR and Rab5 variant Q79L (middle panels) or Rab11 variant S25N (bottom panels). Following serum-deprivation, cells were stimulated with vehicle, 1μM A-61603 or 1mM ISO for 2h. Cells were then fixed and analyzed by confocal microscopy. B . HEK293 cells were transiently transfected with α 1A – AR. After serum deprivation for 24h, cells were pre-treated with a membrane impermeable, disulfide-cleavable biotin reagent to label plasma membrane α 1A – AR. Cells were then left untreated, or stimulated 1 μM A-61603 or 1mM ISO for 5, 30, or 60 min. After treatment, one dish of control cells was harvested without any further manipulations (C: total α 1A – AR). The remaining seven dishes were divided into one control (C+GSH), three treated with A-61603 (A-61603+GSH) and three treated with ISO (ISO+GSH). They were stripped of surface biotin label using a reducing agent, in order to reveal internalized, labeled α 1A – AR. Samples were then analyzed by immunoprecipitation (IP) with streptavidin followed by immunoblotting (IB) with an anti-FLAG antibody.
    Figure Legend Snippet: Stimulation of α 1A -AR transduced HEK-293/EBNA with A-61603 and Iso leads to an increase in intracellular α 1A – AR. A . HEK293 cells were transiently transfected with α 1A – AR only (top panels), or co-transfected with α 1A – AR and Rab5 variant Q79L (middle panels) or Rab11 variant S25N (bottom panels). Following serum-deprivation, cells were stimulated with vehicle, 1μM A-61603 or 1mM ISO for 2h. Cells were then fixed and analyzed by confocal microscopy. B . HEK293 cells were transiently transfected with α 1A – AR. After serum deprivation for 24h, cells were pre-treated with a membrane impermeable, disulfide-cleavable biotin reagent to label plasma membrane α 1A – AR. Cells were then left untreated, or stimulated 1 μM A-61603 or 1mM ISO for 5, 30, or 60 min. After treatment, one dish of control cells was harvested without any further manipulations (C: total α 1A – AR). The remaining seven dishes were divided into one control (C+GSH), three treated with A-61603 (A-61603+GSH) and three treated with ISO (ISO+GSH). They were stripped of surface biotin label using a reducing agent, in order to reveal internalized, labeled α 1A – AR. Samples were then analyzed by immunoprecipitation (IP) with streptavidin followed by immunoblotting (IB) with an anti-FLAG antibody.

    Techniques Used: Transfection, Variant Assay, Confocal Microscopy, Labeling, Immunoprecipitation

    37) Product Images from "Endosomal trafficking of the receptor tyrosine kinase MuSK proceeds via clathrin-dependent pathways, Arf6 and actin"

    Article Title: Endosomal trafficking of the receptor tyrosine kinase MuSK proceeds via clathrin-dependent pathways, Arf6 and actin

    Journal: The Febs Journal

    doi: 10.1111/febs.12309

    Dok7-dependent MuSK activation does not alter the rate of MuSK internalization but increases the recruitment into caveolin-positive structures on the cell surface. (A) To quantify the influence of Dok7 on the rate of MuSK endocytosis, COS-7 cells were transiently transfected with SBP-MuSK alone or together with Dok7. Surface MuSK was labelled with DyLight 649-conjugated streptavidin at 4 °C followed by incubation at 37 °C for different time periods. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. A quantification of internalized MuSK is shown. Error bars indicate the SEM ( n = 3). (B) To determine whether the presence of Dok7 leads to a switch in the endocytic pathway of MuSK, COS-7 cells were transiently transfected with SBP-MuSK together with Dok7. Surface MuSK was labelled with Cy3-conjugated streptavidin at 4 °C followed by incubation at 37 °C for 5 min. After fixation, cells were stained with an antibody against the endogenous clathrin or caveolin. Quantification of colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods) is shown. Error bars indicate the SEM ( n ≥ 12, from at least two independent experiments). (C) To quantify MuSK endocytosis in the presence or absence of inhibitors, COS-7 cells were transiently co-transfected with SBP-MuSK together with Dok7 and, as indicated, with AP180C, Eps15DIII or Dyn2K44A. Cells, treated with dimethylsulfoxide or dynasore, were stained with streptavidin conjugated-DyLight 649 followed by incubation at 37 °C for 5 min. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. Internalized MuSK was quantified. The control sample denotes GFP-positive cells presenting a streptavidin signal. The dimethylsulfoxide sample represents streptavidin-positive cells treated with the solvent dimethylsulfoxide only. Error bars indicate the SEM ( n = 3). (D) COS-7 cells were transiently transfected with SBP-MuSK and GFP or Lrp4-GFP, respectively. MuSK internalization was detected as described in (A). Error bars indicate the SEM (n = 3).
    Figure Legend Snippet: Dok7-dependent MuSK activation does not alter the rate of MuSK internalization but increases the recruitment into caveolin-positive structures on the cell surface. (A) To quantify the influence of Dok7 on the rate of MuSK endocytosis, COS-7 cells were transiently transfected with SBP-MuSK alone or together with Dok7. Surface MuSK was labelled with DyLight 649-conjugated streptavidin at 4 °C followed by incubation at 37 °C for different time periods. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. A quantification of internalized MuSK is shown. Error bars indicate the SEM ( n = 3). (B) To determine whether the presence of Dok7 leads to a switch in the endocytic pathway of MuSK, COS-7 cells were transiently transfected with SBP-MuSK together with Dok7. Surface MuSK was labelled with Cy3-conjugated streptavidin at 4 °C followed by incubation at 37 °C for 5 min. After fixation, cells were stained with an antibody against the endogenous clathrin or caveolin. Quantification of colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods) is shown. Error bars indicate the SEM ( n ≥ 12, from at least two independent experiments). (C) To quantify MuSK endocytosis in the presence or absence of inhibitors, COS-7 cells were transiently co-transfected with SBP-MuSK together with Dok7 and, as indicated, with AP180C, Eps15DIII or Dyn2K44A. Cells, treated with dimethylsulfoxide or dynasore, were stained with streptavidin conjugated-DyLight 649 followed by incubation at 37 °C for 5 min. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. Internalized MuSK was quantified. The control sample denotes GFP-positive cells presenting a streptavidin signal. The dimethylsulfoxide sample represents streptavidin-positive cells treated with the solvent dimethylsulfoxide only. Error bars indicate the SEM ( n = 3). (D) COS-7 cells were transiently transfected with SBP-MuSK and GFP or Lrp4-GFP, respectively. MuSK internalization was detected as described in (A). Error bars indicate the SEM (n = 3).

    Techniques Used: Activation Assay, Transfection, Incubation, Staining, Fluorescence, FACS

    Dok7 expression increases MuSK phosphorylation and its colocalization with phosphotyrosine. (A) MuSK activation by Dok7 was demonstrated by co-expression of SBP-MuSK and HA-tagged Dok7 in COS-7 cells. Surface MuSK was labelled with Cy3-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C. After cell fixation, the cells were stained with an antibody against phosphotyrosine (pTyr, blue) and an antibody against the HA-tag of Dok7 (green). Scale bar = 25 μm. (B) Quantification of MuSK/pTyr colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods) is shown. Error bars indicate the SEM ( n ≥ 10). (C) Quantification of MuSK/Dok7 and MuSK/Dok7/pTyr colocalization during endocytosis (in min) as in (B). Error bars indicate the SEM ( n ≥ 10).
    Figure Legend Snippet: Dok7 expression increases MuSK phosphorylation and its colocalization with phosphotyrosine. (A) MuSK activation by Dok7 was demonstrated by co-expression of SBP-MuSK and HA-tagged Dok7 in COS-7 cells. Surface MuSK was labelled with Cy3-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C. After cell fixation, the cells were stained with an antibody against phosphotyrosine (pTyr, blue) and an antibody against the HA-tag of Dok7 (green). Scale bar = 25 μm. (B) Quantification of MuSK/pTyr colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods) is shown. Error bars indicate the SEM ( n ≥ 10). (C) Quantification of MuSK/Dok7 and MuSK/Dok7/pTyr colocalization during endocytosis (in min) as in (B). Error bars indicate the SEM ( n ≥ 10).

    Techniques Used: Expressing, Activation Assay, Incubation, Staining

    MuSK localizes to structures rich in actin and Arf6. COS-7 cells were transiently transfected with SBP-MuSK. Surface MuSK was labelled with DyLight 649-conjugated streptavidin (blue) at 4 °C followed by incubation at 37 °C for 15 or 60 min. (A) MuSK internalization was visualized in COS-7 cells, which were co-transfected with GFP-tagged actin. The actin cytoskeleton was stained with rhodamine-conjugated phalloidin (red) after cell fixation. (B) MuSK internalization was visualized in COS-7 cells co-transfected with a GFP-tagged Arf6 and stained with phalloidin after cell fixation (red). (C) HeLa cells were transiently transfected with SBP-MuSK together with Arf6-GFP, followed by staining with an antibody against the endogenous Arf6 marker protein MHCI. Magnified structures demonstrating colocalization (arrows) are shown as insets. Scale bar = 25 μm.
    Figure Legend Snippet: MuSK localizes to structures rich in actin and Arf6. COS-7 cells were transiently transfected with SBP-MuSK. Surface MuSK was labelled with DyLight 649-conjugated streptavidin (blue) at 4 °C followed by incubation at 37 °C for 15 or 60 min. (A) MuSK internalization was visualized in COS-7 cells, which were co-transfected with GFP-tagged actin. The actin cytoskeleton was stained with rhodamine-conjugated phalloidin (red) after cell fixation. (B) MuSK internalization was visualized in COS-7 cells co-transfected with a GFP-tagged Arf6 and stained with phalloidin after cell fixation (red). (C) HeLa cells were transiently transfected with SBP-MuSK together with Arf6-GFP, followed by staining with an antibody against the endogenous Arf6 marker protein MHCI. Magnified structures demonstrating colocalization (arrows) are shown as insets. Scale bar = 25 μm.

    Techniques Used: Transfection, Incubation, Staining, Marker

    MuSK internalization proceeds via a clathrin-dependent pathway. (A) To determine whether surface MuSK internalizes via clathrin-or caveolin-positive routes, COS-7 cells were transiently transfected with SBP-MuSK. Surface MuSK was stained with Cy3-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C for 5 min. Endogenous clathrin and caveolin were visualized by antibody staining. MuSK partially colocalizes with these markers (arrowheads in insets). Scale bar = 25 μm. (B) Quantification of MuSK/clathrin, MuSK/caveolin and MuSK/transferrin colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods). Colocalization of MuSK with clathrin and caveolin was analyzed at 0 and 5 min of endocytosis. Transferrin and MuSK colocalization was analyzed at 5 min of endocytosis. The peroxisomal marker PTS2-GFP was used as a negative control. Error bars indicate the SEM ( n ≥ 19; from at least two independent experiments). (C) To determine whether dynamin or clathrin are involved in MuSK internalization, COS-7 cells were either treated with the dynamin specific blocker dynasore or co-transfected with GFP-tagged Dyn2K44A or Myc-tagged AP180C. Surface MuSK was stained with streptavidin conjugated-DyLight 649 (red) at 4 °C followed by incubation at 37 °C for 30 min and subsequent stripping of the remaining surface MuSK molecules. Scale bar = 25 μm. (D) To quantify the blockage of MuSK internalization, COS-7 cells were transiently transfected with SBP-MuSK alone or, as indicated, together with AP180C, Eps15DIII or Dyn2K44A. Cells treated with dimethylsulfoxide (DMSO) or dynasore, were stained with streptavidin-conjugated DyLight 649 followed by incubation at 37 °C for 5 min. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. A quantification of internalized MuSK is shown. The control sample denotes GFP-positive cells presenting a streptavidin signal. The dimethylsulfoxide sample represents streptavidin-positive cells treated with the solvent dimethylsulfoxide only. Error bars indicate the SEM ( n ≥ 5).
    Figure Legend Snippet: MuSK internalization proceeds via a clathrin-dependent pathway. (A) To determine whether surface MuSK internalizes via clathrin-or caveolin-positive routes, COS-7 cells were transiently transfected with SBP-MuSK. Surface MuSK was stained with Cy3-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C for 5 min. Endogenous clathrin and caveolin were visualized by antibody staining. MuSK partially colocalizes with these markers (arrowheads in insets). Scale bar = 25 μm. (B) Quantification of MuSK/clathrin, MuSK/caveolin and MuSK/transferrin colocalization using a threshold-and object-based colocalization analysis (as described in the Materials and methods). Colocalization of MuSK with clathrin and caveolin was analyzed at 0 and 5 min of endocytosis. Transferrin and MuSK colocalization was analyzed at 5 min of endocytosis. The peroxisomal marker PTS2-GFP was used as a negative control. Error bars indicate the SEM ( n ≥ 19; from at least two independent experiments). (C) To determine whether dynamin or clathrin are involved in MuSK internalization, COS-7 cells were either treated with the dynamin specific blocker dynasore or co-transfected with GFP-tagged Dyn2K44A or Myc-tagged AP180C. Surface MuSK was stained with streptavidin conjugated-DyLight 649 (red) at 4 °C followed by incubation at 37 °C for 30 min and subsequent stripping of the remaining surface MuSK molecules. Scale bar = 25 μm. (D) To quantify the blockage of MuSK internalization, COS-7 cells were transiently transfected with SBP-MuSK alone or, as indicated, together with AP180C, Eps15DIII or Dyn2K44A. Cells treated with dimethylsulfoxide (DMSO) or dynasore, were stained with streptavidin-conjugated DyLight 649 followed by incubation at 37 °C for 5 min. The remaining surface staining was stripped off and cells were harvested for intracellular fluorescence detection by FACS. A quantification of internalized MuSK is shown. The control sample denotes GFP-positive cells presenting a streptavidin signal. The dimethylsulfoxide sample represents streptavidin-positive cells treated with the solvent dimethylsulfoxide only. Error bars indicate the SEM ( n ≥ 5).

    Techniques Used: Transfection, Staining, Incubation, Marker, Negative Control, Stripping Membranes, Fluorescence, FACS

    MuSK is transported in Rab7-and Rab4-/11-positive endosomes. (A) To determine whether classical endosomal markers are involved in MuSK trafficking, COS-7 cells were co-transfected with SBP-MuSK together with either GFP-tagged Rab5 (early endosomal compartments, EEA1 positive) or with GFP-tagged Rab7 (late endosomal pathway). Surface MuSK were labelled with DyLight 649-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C for different time periods. Lower panel: quantification of the colocalization of MuSK with Rab5 or Rab7 after different time points using a threshold-and object-based colocalization analysis (as described in the Materials and methods). MuSK/Arf1 was used as a negative control (neg. con). Error bars indicate the SEM ( n ≥ 8). (B) COS-7 cells were co-transfected with SBP-MuSK together with either GFP-tagged Rab4 (early recycling) or with GFP-tagged Rab11 (late recycling). Surface MuSK were labelled with DyLight 649-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C for 60 min. Magnified insets demonstrate a colocalization between MuSK and Rab4 or Rab11. (C) COS-7 cells were transiently transfected with HA-MuSK, stained with an antibody against the extracellular HA-tag followed by incubation at 37 °C for 30 min. The remaining surface antibody staining was stripped off and cells were reincubated at 37 °C for 15 and 30 min (recycling). Cells were fixed and stained with secondary antibodies. Recycled MuSK is detectable at the cell membrane after 15 min. Stripping efficiently removed bound antibodies because no surface MuSK was detectable after 0 min of recycling. Perm, permeabilized. (D) COS-7 cells were co-transfected with either pEGFP, GFP-tagged Rab11 wt or GFP-tagged Rab11 S25N. Surface MuSK was labelled with Cy3-conjugated streptavidin at 4 °C followed by incubation at 37 °C for 120 min. The expression of Rab11 wt increases the recycling of MuSK to the plasma membrane (arrows). Magnified structures are shown as insets. Scale bars = 25 μm.
    Figure Legend Snippet: MuSK is transported in Rab7-and Rab4-/11-positive endosomes. (A) To determine whether classical endosomal markers are involved in MuSK trafficking, COS-7 cells were co-transfected with SBP-MuSK together with either GFP-tagged Rab5 (early endosomal compartments, EEA1 positive) or with GFP-tagged Rab7 (late endosomal pathway). Surface MuSK were labelled with DyLight 649-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C for different time periods. Lower panel: quantification of the colocalization of MuSK with Rab5 or Rab7 after different time points using a threshold-and object-based colocalization analysis (as described in the Materials and methods). MuSK/Arf1 was used as a negative control (neg. con). Error bars indicate the SEM ( n ≥ 8). (B) COS-7 cells were co-transfected with SBP-MuSK together with either GFP-tagged Rab4 (early recycling) or with GFP-tagged Rab11 (late recycling). Surface MuSK were labelled with DyLight 649-conjugated streptavidin (red) at 4 °C followed by incubation at 37 °C for 60 min. Magnified insets demonstrate a colocalization between MuSK and Rab4 or Rab11. (C) COS-7 cells were transiently transfected with HA-MuSK, stained with an antibody against the extracellular HA-tag followed by incubation at 37 °C for 30 min. The remaining surface antibody staining was stripped off and cells were reincubated at 37 °C for 15 and 30 min (recycling). Cells were fixed and stained with secondary antibodies. Recycled MuSK is detectable at the cell membrane after 15 min. Stripping efficiently removed bound antibodies because no surface MuSK was detectable after 0 min of recycling. Perm, permeabilized. (D) COS-7 cells were co-transfected with either pEGFP, GFP-tagged Rab11 wt or GFP-tagged Rab11 S25N. Surface MuSK was labelled with Cy3-conjugated streptavidin at 4 °C followed by incubation at 37 °C for 120 min. The expression of Rab11 wt increases the recycling of MuSK to the plasma membrane (arrows). Magnified structures are shown as insets. Scale bars = 25 μm.

    Techniques Used: Transfection, Incubation, Negative Control, Staining, Stripping Membranes, Expressing

    Blocking Arf6 function disrupts MuSK endocytosis. COS-7 cells were transiently transfected with SBP-MuSK and Arf6-GFP. MuSK was labelled with DyLight 649-conjugated streptavidin (blue) at 4 °C followed by incubation at 37 °C for 60 min. After cell fixation, actin was stained with rhodamine-labeled phalloidin (red). Cells were exposed to different treatments: cytochalasin D, myristoylated Arf6 peptide (myr-Arf6), wortmannin, aluminum fluoride (AlF) or co-transfection with the constitutively active mutant Arf6 Q67L. Dimethylsulfoxide treatment was used as a control. MuSK accumulates in cell protrusions close to the cell membrane. Magnified structures demonstrating colocalization (arrows) are shown as insets. Scale bar = 25 μm.
    Figure Legend Snippet: Blocking Arf6 function disrupts MuSK endocytosis. COS-7 cells were transiently transfected with SBP-MuSK and Arf6-GFP. MuSK was labelled with DyLight 649-conjugated streptavidin (blue) at 4 °C followed by incubation at 37 °C for 60 min. After cell fixation, actin was stained with rhodamine-labeled phalloidin (red). Cells were exposed to different treatments: cytochalasin D, myristoylated Arf6 peptide (myr-Arf6), wortmannin, aluminum fluoride (AlF) or co-transfection with the constitutively active mutant Arf6 Q67L. Dimethylsulfoxide treatment was used as a control. MuSK accumulates in cell protrusions close to the cell membrane. Magnified structures demonstrating colocalization (arrows) are shown as insets. Scale bar = 25 μm.

    Techniques Used: Blocking Assay, Transfection, Incubation, Staining, Labeling, Cotransfection, Mutagenesis

    Visualization of SBP-MuSK endocytosis in a heterologous cell system. (A) COS-7 cells were transiently transfected with SBP-MuSK and cells were stained at 4 °C with streptavidin-conjugated to DyLight 649 (red) followed by incubation at 37 °C for different time points, which allows endocytosis to occur. Newly-synthesized surface MuSK was relabelled with streptavidin-conjugated to DyLight 488 (green). (B) To quantify MuSK endocytosis, transfected COS-7 cells were stained with streptavidin-conjugated to DyLight 649 followed by incubation for different time periods at 37 °C. The remaining surface staining was stripped off. Cells were harvested and internalized MuSK was detected by FACS. A quantification of the detected MuSK signal is shown. t, total surface; s, surface after stripping. Error bars indicate the SEM ( n = 4).
    Figure Legend Snippet: Visualization of SBP-MuSK endocytosis in a heterologous cell system. (A) COS-7 cells were transiently transfected with SBP-MuSK and cells were stained at 4 °C with streptavidin-conjugated to DyLight 649 (red) followed by incubation at 37 °C for different time points, which allows endocytosis to occur. Newly-synthesized surface MuSK was relabelled with streptavidin-conjugated to DyLight 488 (green). (B) To quantify MuSK endocytosis, transfected COS-7 cells were stained with streptavidin-conjugated to DyLight 649 followed by incubation for different time periods at 37 °C. The remaining surface staining was stripped off. Cells were harvested and internalized MuSK was detected by FACS. A quantification of the detected MuSK signal is shown. t, total surface; s, surface after stripping. Error bars indicate the SEM ( n = 4).

    Techniques Used: Transfection, Staining, Incubation, Synthesized, FACS, Stripping Membranes

    38) Product Images from "Proteomic and Functional Analyses of Protein-DNA Complexes During Gene Transfer"

    Article Title: Proteomic and Functional Analyses of Protein-DNA Complexes During Gene Transfer

    Journal: Molecular Therapy

    doi: 10.1038/mt.2012.231

    Proteins are more abundant in pCMV-DTS complexes . Biotinylated plasmids were electroporated into adherent A549 cells, cross-linked at 30 minutes post-transfection, and cells were lysed. Plasmid–protein complexes were isolated via streptavidin-coated
    Figure Legend Snippet: Proteins are more abundant in pCMV-DTS complexes . Biotinylated plasmids were electroporated into adherent A549 cells, cross-linked at 30 minutes post-transfection, and cells were lysed. Plasmid–protein complexes were isolated via streptavidin-coated

    Techniques Used: Transfection, Plasmid Preparation, Isolation

    39) Product Images from "Mutations in Tau Gene Exon 10 Associated with FTDP-17 Alter the Activity of an Exonic Splicing Enhancer to Interact with Tra2β*"

    Article Title: Mutations in Tau Gene Exon 10 Associated with FTDP-17 Alter the Activity of an Exonic Splicing Enhancer to Interact with Tra2β*

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M301800200

    Tra2 β protein interacts with the Tau exon 10 AG-rich region 40 fmol of biotinylated TauEx10−11 RNAs corresponding to dEn, Del280K, WT, and N279K (with zero, one, two, and three copies of AAG repeats, respectively) were incubated on ice for 30 min in 25- μ l reactions under splicing conditions with increasing amounts of cellular extract (0.5 or 1 μ l) containing transiently transfected Myc-tagged Tra2 β ( A ) or HA-tagged SRp40 ( B ). After affinity selection with streptavidin-agarose beads, the bound proteins were detected by Western blotting using anti-Myc antibody (for Tra2 β -Myc) or anti-HA antibody (for SRp40). The intensity of Tra2 β protein pulled down by dEn, Del280K, or N279K RNA in A was quantified, respectively, and compared with that pulled down by WT RNA. The numbers shown below the gel in A represent the relative quantification using the band intensity in the wild type ( lane 5 or lane 6 , respectively) as 1.00 in the presence of either 0.5 μ l ( lanes 1 , 3 , 5 , and 7 ) or 1 μ l ( lanes 2 , 4 , 6 , and 8 ) of Tra2 β -expressing cell lysates.
    Figure Legend Snippet: Tra2 β protein interacts with the Tau exon 10 AG-rich region 40 fmol of biotinylated TauEx10−11 RNAs corresponding to dEn, Del280K, WT, and N279K (with zero, one, two, and three copies of AAG repeats, respectively) were incubated on ice for 30 min in 25- μ l reactions under splicing conditions with increasing amounts of cellular extract (0.5 or 1 μ l) containing transiently transfected Myc-tagged Tra2 β ( A ) or HA-tagged SRp40 ( B ). After affinity selection with streptavidin-agarose beads, the bound proteins were detected by Western blotting using anti-Myc antibody (for Tra2 β -Myc) or anti-HA antibody (for SRp40). The intensity of Tra2 β protein pulled down by dEn, Del280K, or N279K RNA in A was quantified, respectively, and compared with that pulled down by WT RNA. The numbers shown below the gel in A represent the relative quantification using the band intensity in the wild type ( lane 5 or lane 6 , respectively) as 1.00 in the presence of either 0.5 μ l ( lanes 1 , 3 , 5 , and 7 ) or 1 μ l ( lanes 2 , 4 , 6 , and 8 ) of Tra2 β -expressing cell lysates.

    Techniques Used: Incubation, Transfection, Selection, Western Blot, Expressing

    40) Product Images from "CARM1 methylates MED12 to regulate its RNA-binding ability"

    Article Title: CARM1 methylates MED12 to regulate its RNA-binding ability

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800117

    MED12 interacts with TDRD3 in a CARM1-dependent fashion. (A) Fluorograph (top panel) and Coomassie Brilliant Blue staining (bottom panel) of the peptides in vitro methylated by recombinant CARM1 in the presence of tritium-labeled AdoMet. (B) The peptides were used to pull down Tudor domains of the indicated proteins. The input samples and the eluted samples were immunoblotted with αGST antibody. Streptavidin HRP serves as a peptide loading control. (C) MCF-7-Tet-on-shCARM1 cells were untreated or treated with doxycycline (1 μg/ml) for 8 d. Nuclear extracts were subjected to IP with αTDRD3 antibody and the eluted samples were detected by Western blotting with αMED12 and αTDRD3. The input samples were immunoblotted with αTDRD3, αMED12, αmeMED12 a , and αCARM1. (D) HEK293T cells were transiently transfected with FLAG, FLAG-MED12 WT, FLAG-MED12-R1862K, FLAG-MED12-R1899K, and FLAG-MED12-R1862,1899,1912K. Total cell lysates were immunoprecipitated with αTDRD3 antibody and the eluted samples were subjected to Western analysis with αFLAG and αTDRD3 antibodies. The input samples were immunoblotted with αFLAG, αTDRD3, and αβ-actin.
    Figure Legend Snippet: MED12 interacts with TDRD3 in a CARM1-dependent fashion. (A) Fluorograph (top panel) and Coomassie Brilliant Blue staining (bottom panel) of the peptides in vitro methylated by recombinant CARM1 in the presence of tritium-labeled AdoMet. (B) The peptides were used to pull down Tudor domains of the indicated proteins. The input samples and the eluted samples were immunoblotted with αGST antibody. Streptavidin HRP serves as a peptide loading control. (C) MCF-7-Tet-on-shCARM1 cells were untreated or treated with doxycycline (1 μg/ml) for 8 d. Nuclear extracts were subjected to IP with αTDRD3 antibody and the eluted samples were detected by Western blotting with αMED12 and αTDRD3. The input samples were immunoblotted with αTDRD3, αMED12, αmeMED12 a , and αCARM1. (D) HEK293T cells were transiently transfected with FLAG, FLAG-MED12 WT, FLAG-MED12-R1862K, FLAG-MED12-R1899K, and FLAG-MED12-R1862,1899,1912K. Total cell lysates were immunoprecipitated with αTDRD3 antibody and the eluted samples were subjected to Western analysis with αFLAG and αTDRD3 antibodies. The input samples were immunoblotted with αFLAG, αTDRD3, and αβ-actin.

    Techniques Used: Staining, In Vitro, Methylation, Recombinant, Labeling, Western Blot, Transfection, Immunoprecipitation

    41) Product Images from "α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells"

    Article Title: α2,6-hyposialylation of c-Met abolishes cell motility of ST6Gal-I-knockdown HCT116 cells

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2009.84

    c-Met is hyposialylated in ST6Gal-I–null HCT116 cells. (A) ST6Gal-I knockdown in HCT116 cells caused c-Met hyposialylation. Equivalent amounts of cell lysates were incubated with SNA. SNA-glycoprotein complexes were precipitated with streptavidin-conjugated agarose beads and probed by immunoblotting with specific antibody. (B) ST6Gal-I-KD HCT116 cells failed to migrate in response to HGF stimulation. The migration ability of D3, NC, and P cells to HGF stimulation was assessed using a Boyden chamber assay. (C) The migrated cells on the lower surface of the membrane were captured, and five random fields were analyzed using Image-Pro Plus image analysis software. c P
    Figure Legend Snippet: c-Met is hyposialylated in ST6Gal-I–null HCT116 cells. (A) ST6Gal-I knockdown in HCT116 cells caused c-Met hyposialylation. Equivalent amounts of cell lysates were incubated with SNA. SNA-glycoprotein complexes were precipitated with streptavidin-conjugated agarose beads and probed by immunoblotting with specific antibody. (B) ST6Gal-I-KD HCT116 cells failed to migrate in response to HGF stimulation. The migration ability of D3, NC, and P cells to HGF stimulation was assessed using a Boyden chamber assay. (C) The migrated cells on the lower surface of the membrane were captured, and five random fields were analyzed using Image-Pro Plus image analysis software. c P

    Techniques Used: Incubation, Migration, Boyden Chamber Assay, Software

    D3 siRNA transfection does not affect cell surface expression of α2,3-sialic acid structures. Adherent D3, NC, and P cells were first released by trypsinization and then labeled with 2 μg MAA-biotin. After incubation with 0.25 μg RPE-conjugated streptavidin, labeled cells were subjected to FACS analysis. The data are representative of three independent experiments with similar results.
    Figure Legend Snippet: D3 siRNA transfection does not affect cell surface expression of α2,3-sialic acid structures. Adherent D3, NC, and P cells were first released by trypsinization and then labeled with 2 μg MAA-biotin. After incubation with 0.25 μg RPE-conjugated streptavidin, labeled cells were subjected to FACS analysis. The data are representative of three independent experiments with similar results.

    Techniques Used: Transfection, Expressing, Labeling, Incubation, FACS

    42) Product Images from "ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †"

    Article Title: ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.12.5235-5248.2004

    Mapping interacting regions for ABIN-2 on p105 and TPL-2. (A) Schematic diagram of HA-p105 mutants. The relative positions of the Rel homology domain (RHD), ankyrin repeats (ANK), death domain (DD), and PEST region are shown. The N and C termini of the wild-type (WT) HA-p105 protein (amino acids 1 to 968) are indicated. (B) 293 cells were transfected with vectors encoding wild-type (WT) and mutant forms of HA-p105. Cell lysates, prepared using 1% Brij 58 buffer A, were incubated with GST-ABIN-2 1-429 fusion protein or GST (control) coupled to glutathione-Sepharose beads. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) GST-p105 497-968 fusion protein and GST (control) were coupled to glutathione-Sepharose beads and used to affinity purify ABIN-2-FL translated and labeled with [ 35 S]methionine in vitro. Isolated protein was detected by autoradiography of SDS-8% acrylamide gels. (D) 293 cells were transfected with vectors encoding wild-type and mutant forms of Myc-TPL-2. GST-ABIN-2 1-429 was used as an affinity ligand to isolate protein from cell lysates prepared with 1% NP-40 buffer A. Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (E) TPL-2 398-467 peptide coupled to streptavidin-agarose beads was used as an affinity ligand to isolate ABIN-2-FL from lysates of transfected 293 cells. Bound protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting.
    Figure Legend Snippet: Mapping interacting regions for ABIN-2 on p105 and TPL-2. (A) Schematic diagram of HA-p105 mutants. The relative positions of the Rel homology domain (RHD), ankyrin repeats (ANK), death domain (DD), and PEST region are shown. The N and C termini of the wild-type (WT) HA-p105 protein (amino acids 1 to 968) are indicated. (B) 293 cells were transfected with vectors encoding wild-type (WT) and mutant forms of HA-p105. Cell lysates, prepared using 1% Brij 58 buffer A, were incubated with GST-ABIN-2 1-429 fusion protein or GST (control) coupled to glutathione-Sepharose beads. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) GST-p105 497-968 fusion protein and GST (control) were coupled to glutathione-Sepharose beads and used to affinity purify ABIN-2-FL translated and labeled with [ 35 S]methionine in vitro. Isolated protein was detected by autoradiography of SDS-8% acrylamide gels. (D) 293 cells were transfected with vectors encoding wild-type and mutant forms of Myc-TPL-2. GST-ABIN-2 1-429 was used as an affinity ligand to isolate protein from cell lysates prepared with 1% NP-40 buffer A. Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (E) TPL-2 398-467 peptide coupled to streptavidin-agarose beads was used as an affinity ligand to isolate ABIN-2-FL from lysates of transfected 293 cells. Bound protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting.

    Techniques Used: Transfection, Mutagenesis, Incubation, Affinity Purification, SDS Page, Western Blot, Labeling, In Vitro, Isolation, Autoradiography

    43) Product Images from "Role of IGF-1R in ameliorating apoptosis of GNE deficient cells"

    Article Title: Role of IGF-1R in ameliorating apoptosis of GNE deficient cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25510-9

    Determination of IGF-1R sialylation levels using Lectin affinity assay: ( A ) Sialylation status of IGF-1R: Equal amount of cell lysates from vector control cells were treated with 100 mU of C. perfringen s sialidase and untreated lysates from GNE knockdown cells were separated on 8% SDS-PAGE followed by immunoblotting with anti-α IGF-1R. ( B ) SNA Lectin affinity assay: Sialylated IGF-1R from GNE knockdown cells and scrambled shRNA vector control cell was pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. ( C ) Sialylated IGF-1R complexes from GNE mutant and pcDNA3 transfected vector control cells were pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. Sialylated IGF-1R complexes from 100 mU neuraminidase treated vector control cells that were pulled down using biotin labelled SNA/streptavidin-coupled agarose served as negative control in this experiment. Equal amount of protein was used as input lysates. ( D – F ) are showing representative densitometry graphs of ( A – C ), respectively, normalized to vector control.
    Figure Legend Snippet: Determination of IGF-1R sialylation levels using Lectin affinity assay: ( A ) Sialylation status of IGF-1R: Equal amount of cell lysates from vector control cells were treated with 100 mU of C. perfringen s sialidase and untreated lysates from GNE knockdown cells were separated on 8% SDS-PAGE followed by immunoblotting with anti-α IGF-1R. ( B ) SNA Lectin affinity assay: Sialylated IGF-1R from GNE knockdown cells and scrambled shRNA vector control cell was pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. ( C ) Sialylated IGF-1R complexes from GNE mutant and pcDNA3 transfected vector control cells were pulled down using biotin labelled SNA/streptavidin-coupled agarose and immunoblotted with anti-α IGF-1R. Sialylated IGF-1R complexes from 100 mU neuraminidase treated vector control cells that were pulled down using biotin labelled SNA/streptavidin-coupled agarose served as negative control in this experiment. Equal amount of protein was used as input lysates. ( D – F ) are showing representative densitometry graphs of ( A – C ), respectively, normalized to vector control.

    Techniques Used: Plasmid Preparation, SDS Page, shRNA, Mutagenesis, Transfection, Negative Control

    44) Product Images from "TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis"

    Article Title: TP53TG1 enhances cisplatin sensitivity of non-small cell lung cancer cells through regulating miR-18a/PTEN axis

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-018-0221-7

    TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P
    Figure Legend Snippet: TP53TG1 inhibited miR-18a expression in NSCLC cells. a Sequence alignment of miR-18a with the putative binding sites within the wild-type regions of TP53TG1. b Subcellular fractionation assay was performed to identify the subcellular location of TP53TG1 with GAPDH and U6 as internal references. c , d The luciferase activity was detected in A549 cells transfected with TP53TG1-WT or TP53TG1-MUT and miR-con, miR-18a mimics, anti-miR-con or anti-miR-18a. e Biotin-labeled TP53TG1 RNA was obtained and added to cell lysates with Streptavidin agarose beads, followed by the detection of miR-18a enrichment by RNA pull-down assay. f RIP assay was performed to evaluate the endogenous binding between TP53TG1 and miR-18a in A549 cells using specific antibody against Ago2, followed by detection of RNA levels by qRT-PCR. g qRT-PCR assay of miR-18a expression in A549 cells transfected with si-TP53TG1#1 or pcDNA-TP53TG1 for 48 h. h qRT-PCR assay of miR-18a expression in 40 pairs of NSCLC samples. i qRT-PCR assay of miR-18a expression in DDP-sensitive NSCLC tissues and DDP-resistant NSCLC samples. j The correlation between TP53TG1 and miR-18a expression was detected in NSCLC samples. All experiments are repeated three times. * P

    Techniques Used: Expressing, Sequencing, Binding Assay, Fractionation, Luciferase, Activity Assay, Transfection, Labeling, Pull Down Assay, Quantitative RT-PCR

    45) Product Images from "Intrinsic Disorder in Transmembrane Proteins: Roles in Signaling and Topology Prediction"

    Article Title: Intrinsic Disorder in Transmembrane Proteins: Roles in Signaling and Topology Prediction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158594

    Topology prediction of a multi-pass transmembrane protein according to the localization of its IDRs. (A) Topology prediction of TMEM117 (UniProtID: Q9H0C3) according to the localization of its C-terminal IDRs, with the IN label describing the cytoplasmic part of the protein and the OUT labels the extracellular part. The blue dots represent the average disorder score using PONDR-FIT, IUPRED and DISOPRED2, and the error bars the standard error. The blue lanes show the position of the transmembrane domains. (B) Immunofluorescence of HeLa transiently expressing TMEM117-V5. Cells were fixed, permeabilized and stained for TMEM117-V5 and CLIMP63 (UniProtID: Q07065) for Endoplasmic Reticulum visualization. (C) Surface biotinylation of HeLa transiently expressing TMEM117-V5. Plasma membrane proteins were labelled with biotin, immunoprecipitated by streptavidin conjugated beads and probed by western blot against V5, transferrin receptor and GAPDH. The total cell extract (TCE) represents 10% of the immunoprecipitation volume. (D) TMEM117-V5 was immunoprecipitated with an anti V5 antibody from extracts of HeLa transiently expressing the protein. The precipitate was then left untreated or treated with N-Glycosidase F or EndoH and the effect of the treatment analyzed by SDS-PAGE and western blotting against the V5 tag. * aspecific band. (E) Expression of TMEM117 glycosylation mutants in HeLa. Cells were transfected for 48h and the wild-type and mutant proteins were immunoprecipitated using a mouse anti V5 monoclonal antibody and subsequently analyzed by SDS-PAGE and western blotting using a rabbit anti V5 antibody. (F) Immunofluorescence on HeLa transiently expressing TMEM117-GFP (green signal). Cells were fixed in 4% PFA and left non permeabilized or permeabilized with 0.1% Triton X100. Cells were then stained with a mouse anti-GFP primary antibody coupled to an Alexa 568 anti-mouse secondary antibody (red signal) and Hoechst for the nuclei staining in both conditions. (G) Cartoon representing the experimentally observed topology of TMEM117, the localization of the two N-Glycosylation sites and the GFP or V5 tags. For (C, D and E) n.t. = mock transfected controls.
    Figure Legend Snippet: Topology prediction of a multi-pass transmembrane protein according to the localization of its IDRs. (A) Topology prediction of TMEM117 (UniProtID: Q9H0C3) according to the localization of its C-terminal IDRs, with the IN label describing the cytoplasmic part of the protein and the OUT labels the extracellular part. The blue dots represent the average disorder score using PONDR-FIT, IUPRED and DISOPRED2, and the error bars the standard error. The blue lanes show the position of the transmembrane domains. (B) Immunofluorescence of HeLa transiently expressing TMEM117-V5. Cells were fixed, permeabilized and stained for TMEM117-V5 and CLIMP63 (UniProtID: Q07065) for Endoplasmic Reticulum visualization. (C) Surface biotinylation of HeLa transiently expressing TMEM117-V5. Plasma membrane proteins were labelled with biotin, immunoprecipitated by streptavidin conjugated beads and probed by western blot against V5, transferrin receptor and GAPDH. The total cell extract (TCE) represents 10% of the immunoprecipitation volume. (D) TMEM117-V5 was immunoprecipitated with an anti V5 antibody from extracts of HeLa transiently expressing the protein. The precipitate was then left untreated or treated with N-Glycosidase F or EndoH and the effect of the treatment analyzed by SDS-PAGE and western blotting against the V5 tag. * aspecific band. (E) Expression of TMEM117 glycosylation mutants in HeLa. Cells were transfected for 48h and the wild-type and mutant proteins were immunoprecipitated using a mouse anti V5 monoclonal antibody and subsequently analyzed by SDS-PAGE and western blotting using a rabbit anti V5 antibody. (F) Immunofluorescence on HeLa transiently expressing TMEM117-GFP (green signal). Cells were fixed in 4% PFA and left non permeabilized or permeabilized with 0.1% Triton X100. Cells were then stained with a mouse anti-GFP primary antibody coupled to an Alexa 568 anti-mouse secondary antibody (red signal) and Hoechst for the nuclei staining in both conditions. (G) Cartoon representing the experimentally observed topology of TMEM117, the localization of the two N-Glycosylation sites and the GFP or V5 tags. For (C, D and E) n.t. = mock transfected controls.

    Techniques Used: Immunofluorescence, Expressing, Staining, Immunoprecipitation, Western Blot, SDS Page, Transfection, Mutagenesis

    46) Product Images from "Identification and characterization of RNA guanine-quadruplex binding proteins"

    Article Title: Identification and characterization of RNA guanine-quadruplex binding proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku290

    Pull-down assays. ( A ) and ( B ) G-quadruplex motifs of the MMP16 (A) and ARPC2 (B) mRNAs were coupled to streptavidin agarose beads. Whole-cell extract of HEK293 cells was added to the beads. After several washing steps, proteins were eluted with buffers containing the indicated K + -concentrations. Proteins were analyzed by SDS-PAGE. Proteins that bind exclusively to the G-quadruplex-sequence (Q) are indicated. GQ-M represents an independent experiment, in which the HEK293 extract was first incubated with the mutated oligonucleotide and then with the G-quadruplex-forming RNA. The indicated proteins were identified by MS-based peptide mass fingerprinting. ( C ) Pull-down assay with the additional ARPC2 control oligonucleotide with two G-stretches (ARPC2–2xG). 1. ME2 (66 kDa); 2. YB-1 (36 kDa); 3. U2AF65 (54 kDa); 4. hnRNPH (50 kDa); 5. YB-1 (36 kDa) hnRNPF (46 kDa); 6. RPS2 (32 kDa); 7. Nucl (76 kDa); 8. RBM14 (70 kDa); 9. SRSF1 (28 kDa); 10. SRSF1 (28 kDa) RPS6 (29 kDa); 11. RPL7 (29 kDa); 12. SRSF9 (26 kDa), RPS6 (29 kDa); 13. SRSF9 (26 kDa), RPL14 (23,5 kDa); 14. RPL10 (25 kDa) RPS9 (23 kDa); 15. RPL26 (17 kDa); 16. RPL27a (17 kDa); 17. RPS9 (23 kDa); a. Actin (42 kDa); b. hnRNPA3 (39 kDa); c. hnRNPA2B1 (37 kDa) and hnRNPA3 (39 kDa); d. hnRNPA2B1 (37 kDa); e. hnRNPA1 (39 kDa); f. YB-1 (36 kDA); g. YB-1/Actin (36 kDa, 42 kDa).
    Figure Legend Snippet: Pull-down assays. ( A ) and ( B ) G-quadruplex motifs of the MMP16 (A) and ARPC2 (B) mRNAs were coupled to streptavidin agarose beads. Whole-cell extract of HEK293 cells was added to the beads. After several washing steps, proteins were eluted with buffers containing the indicated K + -concentrations. Proteins were analyzed by SDS-PAGE. Proteins that bind exclusively to the G-quadruplex-sequence (Q) are indicated. GQ-M represents an independent experiment, in which the HEK293 extract was first incubated with the mutated oligonucleotide and then with the G-quadruplex-forming RNA. The indicated proteins were identified by MS-based peptide mass fingerprinting. ( C ) Pull-down assay with the additional ARPC2 control oligonucleotide with two G-stretches (ARPC2–2xG). 1. ME2 (66 kDa); 2. YB-1 (36 kDa); 3. U2AF65 (54 kDa); 4. hnRNPH (50 kDa); 5. YB-1 (36 kDa) hnRNPF (46 kDa); 6. RPS2 (32 kDa); 7. Nucl (76 kDa); 8. RBM14 (70 kDa); 9. SRSF1 (28 kDa); 10. SRSF1 (28 kDa) RPS6 (29 kDa); 11. RPL7 (29 kDa); 12. SRSF9 (26 kDa), RPS6 (29 kDa); 13. SRSF9 (26 kDa), RPL14 (23,5 kDa); 14. RPL10 (25 kDa) RPS9 (23 kDa); 15. RPL26 (17 kDa); 16. RPL27a (17 kDa); 17. RPS9 (23 kDa); a. Actin (42 kDa); b. hnRNPA3 (39 kDa); c. hnRNPA2B1 (37 kDa) and hnRNPA3 (39 kDa); d. hnRNPA2B1 (37 kDa); e. hnRNPA1 (39 kDa); f. YB-1 (36 kDA); g. YB-1/Actin (36 kDa, 42 kDa).

    Techniques Used: SDS Page, Sequencing, Incubation, Mass Spectrometry, Peptide Mass Fingerprinting, Pull Down Assay

    47) Product Images from "Age-dependent B cell Autoimmunity to a Myelin Surface Antigen in Pediatric Multiple Sclerosis"

    Article Title: Age-dependent B cell Autoimmunity to a Myelin Surface Antigen in Pediatric Multiple Sclerosis

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0801888

    Antibodies in pediatric MS sera are specific for MOG A. A monoclonal antibody to MOG competes with serum antibodies for antigen binding. Sera and indicated amounts of the MOG-specific antibody 8–18C5 were incubated together with MOG-GFP cells for one hour. A human-specific secondary antibody was used to detect bound IgG. Addition of increasing amounts of 8–18C5 degreased the amount of serum antibody bound to cells. B. Affinity-purified antibodies specifically bind to MOG transfectants. Biotinylated recombinant MOG (rMOG, extracellular domain) or an Ig superfamily control protein (membrane-proximal Ig domain of CD80) were captured onto streptavidin beads for affinity isolation of MOG-specific antibodies from 5µL of serum. Unbound and eluted antibodies were used to stain MOG and control transfectants. Binding ratios obtained with purified antibodies from four sera are summarized in the table. Antibodies purified on MOG beads bound MOG on the cell surface, and addition of 25µg soluble recombinant MOG inhibited binding of eluted MOG-specific antibodies for all four sera tested.
    Figure Legend Snippet: Antibodies in pediatric MS sera are specific for MOG A. A monoclonal antibody to MOG competes with serum antibodies for antigen binding. Sera and indicated amounts of the MOG-specific antibody 8–18C5 were incubated together with MOG-GFP cells for one hour. A human-specific secondary antibody was used to detect bound IgG. Addition of increasing amounts of 8–18C5 degreased the amount of serum antibody bound to cells. B. Affinity-purified antibodies specifically bind to MOG transfectants. Biotinylated recombinant MOG (rMOG, extracellular domain) or an Ig superfamily control protein (membrane-proximal Ig domain of CD80) were captured onto streptavidin beads for affinity isolation of MOG-specific antibodies from 5µL of serum. Unbound and eluted antibodies were used to stain MOG and control transfectants. Binding ratios obtained with purified antibodies from four sera are summarized in the table. Antibodies purified on MOG beads bound MOG on the cell surface, and addition of 25µg soluble recombinant MOG inhibited binding of eluted MOG-specific antibodies for all four sera tested.

    Techniques Used: Mass Spectrometry, Binding Assay, Incubation, Affinity Purification, Recombinant, Isolation, Staining, Purification

    Antibodies to other myelin surface proteins in pediatric MS sera A. Generation of transfectants that express other myelin proteins on the cell surface. Jurkat cells were transfected with vectors that drove expression of zsGreen as well as MOG, myelin-associated glycoprotein (MAG) or oligodendrocyte-myelin glycoprotein (OMG). Transfectants were cloned by single cell sorting and surface expression was verified using an antibody to the HA tag attached to the N-terminus of each protein. B. Examples of labeling of these transfectants with MOG-GFP positive pediatric MS sera. Samples were incubated at a 1:50 dilution with Jurkat cells transfected with HA-tagged antigens, and bound IgG was detected with biotinylated anti-human IgG and streptavidin-PE. C. Pediatric MS sera with MOG antibodies do not show broad anti-myelin reactivity. Antibody binding for 13 serum samples is shown as the median fluorescence intensity of PE. Although antibodies to MAG and OMG were detectable at low levels in a few sera, the labeling of MOG transfectants was considerably brighter in all cases. Antibodies to OMG and MAG were not detected in MOG negative pediatric MS sera or pediatric controls (not shown).
    Figure Legend Snippet: Antibodies to other myelin surface proteins in pediatric MS sera A. Generation of transfectants that express other myelin proteins on the cell surface. Jurkat cells were transfected with vectors that drove expression of zsGreen as well as MOG, myelin-associated glycoprotein (MAG) or oligodendrocyte-myelin glycoprotein (OMG). Transfectants were cloned by single cell sorting and surface expression was verified using an antibody to the HA tag attached to the N-terminus of each protein. B. Examples of labeling of these transfectants with MOG-GFP positive pediatric MS sera. Samples were incubated at a 1:50 dilution with Jurkat cells transfected with HA-tagged antigens, and bound IgG was detected with biotinylated anti-human IgG and streptavidin-PE. C. Pediatric MS sera with MOG antibodies do not show broad anti-myelin reactivity. Antibody binding for 13 serum samples is shown as the median fluorescence intensity of PE. Although antibodies to MAG and OMG were detectable at low levels in a few sera, the labeling of MOG transfectants was considerably brighter in all cases. Antibodies to OMG and MAG were not detected in MOG negative pediatric MS sera or pediatric controls (not shown).

    Techniques Used: Mass Spectrometry, Transfection, Expressing, Clone Assay, FACS, Labeling, Incubation, Binding Assay, Fluorescence

    Identification of autoantibodies to native MOG in pediatric MS patients A. Brightness of fluorescent labeling with different positive pediatric MS sera. FACS data representing antibody binding to the MOG-GFP transfectant (open histograms) and the control GFP transfectant (shaded histograms) are shown for sera from six pediatric MS patients. Sera were incubated at a dilution of 1:50 with cells, and bound antibodies were detected using a biotinylated anti-human IgG and streptavidin-PE. For each sample, the ratio of the mean fluorescence intensity for the MOG and the control transfectant is shown in the top left corner. The six depicted examples represent the entire range of fluorescent intensities for positive sera, with binding ratios of approximately 5 to 200 for the MOG versus the control transfectant. B. Antibody titers measured by FACS analysis. Sera were incubated with MOG-GFP cells at the indicated dilutions. Antibodies to MOG were detectable in all positive sera at a dilution of 1:400 and many at a dilution of 1:800.
    Figure Legend Snippet: Identification of autoantibodies to native MOG in pediatric MS patients A. Brightness of fluorescent labeling with different positive pediatric MS sera. FACS data representing antibody binding to the MOG-GFP transfectant (open histograms) and the control GFP transfectant (shaded histograms) are shown for sera from six pediatric MS patients. Sera were incubated at a dilution of 1:50 with cells, and bound antibodies were detected using a biotinylated anti-human IgG and streptavidin-PE. For each sample, the ratio of the mean fluorescence intensity for the MOG and the control transfectant is shown in the top left corner. The six depicted examples represent the entire range of fluorescent intensities for positive sera, with binding ratios of approximately 5 to 200 for the MOG versus the control transfectant. B. Antibody titers measured by FACS analysis. Sera were incubated with MOG-GFP cells at the indicated dilutions. Antibodies to MOG were detectable in all positive sera at a dilution of 1:400 and many at a dilution of 1:800.

    Techniques Used: Mass Spectrometry, Labeling, FACS, Binding Assay, Transfection, Incubation, Fluorescence

    48) Product Images from "A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning"

    Article Title: A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning

    Journal: eLife

    doi: 10.7554/eLife.11466

    Identification of potential CRM1 cargoes from 3 species. ( A ) Mouse (mm) or yeast (sc) CRM1 were immobilised through a biotinylated Avi-tag to streptavidin agarose, and incubated with indicated extracts (1 ml), without or with the addition of 5 μM RanQ69L 5-180 GTP. The beads were thoroughly washed and subsequently eluted at 45°C with SDS sample buffer (which leaves the biotin-streptavidin interaction largely intact). Analysis of indicated samples was by SDS-PAGE and Coomassie-staining. 1/200 of the starting extracts and 1/5 of eluates were loaded. ( B ) Starting extracts, CRM1 w/o Ran, and CRM1+RanGTP samples were analysed by mass spectrometry. Venn diagrams represent identified unique proteins. Numbers in parenthesis include also proteins that were not identified in a total Xenopus extract or the ‘CRM1+RanGTP’ sample, but in the isolated nuclear and cytoplasmic fractions; these proteins extend the list of ‘CRM1-non-binders’. DOI: http://dx.doi.org/10.7554/eLife.11466.005
    Figure Legend Snippet: Identification of potential CRM1 cargoes from 3 species. ( A ) Mouse (mm) or yeast (sc) CRM1 were immobilised through a biotinylated Avi-tag to streptavidin agarose, and incubated with indicated extracts (1 ml), without or with the addition of 5 μM RanQ69L 5-180 GTP. The beads were thoroughly washed and subsequently eluted at 45°C with SDS sample buffer (which leaves the biotin-streptavidin interaction largely intact). Analysis of indicated samples was by SDS-PAGE and Coomassie-staining. 1/200 of the starting extracts and 1/5 of eluates were loaded. ( B ) Starting extracts, CRM1 w/o Ran, and CRM1+RanGTP samples were analysed by mass spectrometry. Venn diagrams represent identified unique proteins. Numbers in parenthesis include also proteins that were not identified in a total Xenopus extract or the ‘CRM1+RanGTP’ sample, but in the isolated nuclear and cytoplasmic fractions; these proteins extend the list of ‘CRM1-non-binders’. DOI: http://dx.doi.org/10.7554/eLife.11466.005

    Techniques Used: Incubation, SDS Page, Staining, Mass Spectrometry, Isolation

    49) Product Images from "?- but not ?-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia"

    Article Title: ?- but not ?-secretase proteolysis of APP causes synaptic and memory deficits in a mouse model of dementia

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201100195

    A BRI2-derived peptide binds APP and inhibits β-cleavage of APP A-B. HEK293-APP cells were incubated with the indicated peptides. β- and α-cleavage of APP were quantified by measuring sAPPβ and sAPPα in media by WB. WB of cell lysates detected APP and α-Tubulin. C. WB analysis of cell lysates and conditioned media from HEK293-APP cells treated either with the indicated concentrations of either N3-2A or N1. In a duplicate experiment, cells were treated with compound-E (+) while incubated with the indicated peptides. Lysates were probed for APP, APP-CTFs and α-Tubulin, culture media was probed for sAPPα and sAPPβ. In the right panel, the results of a similar experiment in HeLa-APP cells are shown. D-F. WB analysis of lysates (L) or α-Flag IP (IP) from HeLa/APP cells incubated for 2 h with Flag-tagged peptides. D. Cells were incubated at either 37 or 4 °C with or without 40 µM N3-2A-F. E. The indicated concentrations of N3-2A were added to the media containing 40 µM N3-2A-F. F. Cells were incubated with 40 µM N3-2A-F, N4-F or N3-4A-F. G. Brain cells were cultured as in ( D ). H. Biotinylated cells were cultured as in ( D ). The reduced and not reduced samples are indicated (+red and −red, respectively). Lysates (L), α-Flag IP eluted with Flag-peptide (E), eluted sample precipitated with streptavidin-beads [both the fraction unbound (U) and bound (B) to streptavidin-beads], were probed for APP in WB. I. Purified β-secretase was incubated with fluorescent β-secretase substrate for 30 min, resulting in β-cleavage that could be detected by fluorescence increase. In separate samples, the indicated concentrations of N3-2A or β-secretase-inhibitor IV were added to the reaction. The data are shown as % of inhibition of β-secretase activity in samples without inhibitors. J. Model depicting the mechanism of action of N3-2A/MoBA. The peptide interferes with processing of APP by β-secretase but, unlike full-length BRI2, does not modulate γ-cleavage of β-CTF.
    Figure Legend Snippet: A BRI2-derived peptide binds APP and inhibits β-cleavage of APP A-B. HEK293-APP cells were incubated with the indicated peptides. β- and α-cleavage of APP were quantified by measuring sAPPβ and sAPPα in media by WB. WB of cell lysates detected APP and α-Tubulin. C. WB analysis of cell lysates and conditioned media from HEK293-APP cells treated either with the indicated concentrations of either N3-2A or N1. In a duplicate experiment, cells were treated with compound-E (+) while incubated with the indicated peptides. Lysates were probed for APP, APP-CTFs and α-Tubulin, culture media was probed for sAPPα and sAPPβ. In the right panel, the results of a similar experiment in HeLa-APP cells are shown. D-F. WB analysis of lysates (L) or α-Flag IP (IP) from HeLa/APP cells incubated for 2 h with Flag-tagged peptides. D. Cells were incubated at either 37 or 4 °C with or without 40 µM N3-2A-F. E. The indicated concentrations of N3-2A were added to the media containing 40 µM N3-2A-F. F. Cells were incubated with 40 µM N3-2A-F, N4-F or N3-4A-F. G. Brain cells were cultured as in ( D ). H. Biotinylated cells were cultured as in ( D ). The reduced and not reduced samples are indicated (+red and −red, respectively). Lysates (L), α-Flag IP eluted with Flag-peptide (E), eluted sample precipitated with streptavidin-beads [both the fraction unbound (U) and bound (B) to streptavidin-beads], were probed for APP in WB. I. Purified β-secretase was incubated with fluorescent β-secretase substrate for 30 min, resulting in β-cleavage that could be detected by fluorescence increase. In separate samples, the indicated concentrations of N3-2A or β-secretase-inhibitor IV were added to the reaction. The data are shown as % of inhibition of β-secretase activity in samples without inhibitors. J. Model depicting the mechanism of action of N3-2A/MoBA. The peptide interferes with processing of APP by β-secretase but, unlike full-length BRI2, does not modulate γ-cleavage of β-CTF.

    Techniques Used: Derivative Assay, Incubation, Western Blot, Cell Culture, Purification, Fluorescence, Inhibition, Activity Assay

    50) Product Images from "Arsenite Targets the Zinc Finger Domains of Tet Proteins and Inhibits Tet-Mediated Oxidation of 5-Methylcytosine"

    Article Title: Arsenite Targets the Zinc Finger Domains of Tet Proteins and Inhibits Tet-Mediated Oxidation of 5-Methylcytosine

    Journal: Environmental science & technology

    doi: 10.1021/acs.est.5b03386

    Binding behavior between NaAsO 2 and human Tet proteins. (a) Higher-resolution “ultra-zoom-scan” ESI–MS showing the [M + 2H] 2+ ions of a synthetic peptide derived from human Tet2 in the absence or presence of As(III). (b) UV absorption spectra of the synthetic Tet2 peptide titrated with increasing concentrations of arsenite. (c) Streptavidin agarose affinity pull-down assay using biotin-As as a probe revealed the binding between As(III) and Tet1 in cells.
    Figure Legend Snippet: Binding behavior between NaAsO 2 and human Tet proteins. (a) Higher-resolution “ultra-zoom-scan” ESI–MS showing the [M + 2H] 2+ ions of a synthetic peptide derived from human Tet2 in the absence or presence of As(III). (b) UV absorption spectra of the synthetic Tet2 peptide titrated with increasing concentrations of arsenite. (c) Streptavidin agarose affinity pull-down assay using biotin-As as a probe revealed the binding between As(III) and Tet1 in cells.

    Techniques Used: Binding Assay, Mass Spectrometry, Derivative Assay, Pull Down Assay

    51) Product Images from "Keratin 23 promotes telomerase reverse transcriptase expression and human colorectal cancer growth"

    Article Title: Keratin 23 promotes telomerase reverse transcriptase expression and human colorectal cancer growth

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.339

    KRT23 was identified and validated as a transcription factor of hTERT in CRC cells. ( a ) A streptavidin–biotin pulldown assay was performed to identify the specific proteins that bind to the hTERT promoter. Nuclear extracts prepared from human CRC cells (SW620, RKO, DLD1 and LoVo) and hepatoma carcinoma cells (SNU, HEPG2 and BEL) were incubated with a biotin-labeled hTERT promoter probe and streptavidin–agarose beads. The DNA–protein complexes were separated by SDS-PAGE, and the protein bands were visualized by silver staining. The protein bands (at ~35 kDa) were excised from the gel and identified by the mass spectrum analysis. We predicted that the candidate CRC-specific hTERT promoter-binding protein was KRT23. ( b ) Immunoblot assay for detecting KRT23 binding to the hTERT promoter probe. KRT23 protein in the DNA–protein complexes was detected by western blot assay using an anti-KRT23 antibody. ( c ) Chromatin immunoprecipitation (CHIP) assays were performed using the hTERT promoter from human CRC cells (SW620, RKO, DLD1 and LoVo). PCR products were separated on 1% agarose gels. Normal immunoglobulin G (IgG) was the negative control for the KRT23 antibody
    Figure Legend Snippet: KRT23 was identified and validated as a transcription factor of hTERT in CRC cells. ( a ) A streptavidin–biotin pulldown assay was performed to identify the specific proteins that bind to the hTERT promoter. Nuclear extracts prepared from human CRC cells (SW620, RKO, DLD1 and LoVo) and hepatoma carcinoma cells (SNU, HEPG2 and BEL) were incubated with a biotin-labeled hTERT promoter probe and streptavidin–agarose beads. The DNA–protein complexes were separated by SDS-PAGE, and the protein bands were visualized by silver staining. The protein bands (at ~35 kDa) were excised from the gel and identified by the mass spectrum analysis. We predicted that the candidate CRC-specific hTERT promoter-binding protein was KRT23. ( b ) Immunoblot assay for detecting KRT23 binding to the hTERT promoter probe. KRT23 protein in the DNA–protein complexes was detected by western blot assay using an anti-KRT23 antibody. ( c ) Chromatin immunoprecipitation (CHIP) assays were performed using the hTERT promoter from human CRC cells (SW620, RKO, DLD1 and LoVo). PCR products were separated on 1% agarose gels. Normal immunoglobulin G (IgG) was the negative control for the KRT23 antibody

    Techniques Used: Incubation, Labeling, SDS Page, Silver Staining, Binding Assay, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control

    52) Product Images from "Identification of novel proteins associated with yeast snR30 small nucleolar RNA"

    Article Title: Identification of novel proteins associated with yeast snR30 small nucleolar RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr659

    Selective purification of S1-tagged snR30 snoRNP. One-step (A and B) or two-step (C) purifications were done with extracts prepared from the S1-SNR30 strain grown in YPD. ( A ) 3′-End labeling analysis of recovered RNAs. Labeled RNAs from the extract (T), Gar1-TAP IP and S1-snR30 purification were analyzed. Radiolabeled DNA markers (M) were run in parallel. S1-snR30, H/ACA snoRNAs, 5.8S and 5S rRNAs, and tRNAs are indicated. ( B ) Northern blots of RNAs recovered from one-step purification: extract (T), streptavidin flow through (S) and eluate (P). ( C ) Northern blots of RNAs recovered from two-step purification: extract (T), IgG flow through (S1), IgG eluate (P1) and streptavidin eluate (P2). For B and C, snoRNAs analyzed are indicated on the right of each panel.
    Figure Legend Snippet: Selective purification of S1-tagged snR30 snoRNP. One-step (A and B) or two-step (C) purifications were done with extracts prepared from the S1-SNR30 strain grown in YPD. ( A ) 3′-End labeling analysis of recovered RNAs. Labeled RNAs from the extract (T), Gar1-TAP IP and S1-snR30 purification were analyzed. Radiolabeled DNA markers (M) were run in parallel. S1-snR30, H/ACA snoRNAs, 5.8S and 5S rRNAs, and tRNAs are indicated. ( B ) Northern blots of RNAs recovered from one-step purification: extract (T), streptavidin flow through (S) and eluate (P). ( C ) Northern blots of RNAs recovered from two-step purification: extract (T), IgG flow through (S1), IgG eluate (P1) and streptavidin eluate (P2). For B and C, snoRNAs analyzed are indicated on the right of each panel.

    Techniques Used: Purification, End Labeling, Labeling, Northern Blot, Flow Cytometry

    Purified S1-snR30 contains H/ACA proteins and a number of additional proteins. Proteins present in the extract (T), IgG eluate (P1) and streptavidin eluate (P2) were separated by SDS–PAGE and subjected to immunoblotting to detect Cbf5 and Nhp2 ( A ). In ( B ), the Criterion XT gel was silver-stained. Proteins identified by MS are indicated on the right. The molecular weights of protein markers (lane 1) are indicated on the left in kDa.
    Figure Legend Snippet: Purified S1-snR30 contains H/ACA proteins and a number of additional proteins. Proteins present in the extract (T), IgG eluate (P1) and streptavidin eluate (P2) were separated by SDS–PAGE and subjected to immunoblotting to detect Cbf5 and Nhp2 ( A ). In ( B ), the Criterion XT gel was silver-stained. Proteins identified by MS are indicated on the right. The molecular weights of protein markers (lane 1) are indicated on the left in kDa.

    Techniques Used: Purification, SDS Page, Staining, Mass Spectrometry

    53) Product Images from "Assembly with the NR1 Subunit Is Required for Surface Expression of NR3A-Containing NMDA Receptors"

    Article Title: Assembly with the NR1 Subunit Is Required for Surface Expression of NR3A-Containing NMDA Receptors

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-04-01228.2001

    NR3A is present at the cell surface only when coexpressed with the NR1-1a subunit. A , HEK293T cells were transfected with different combinations of NMDA receptor subunits and incubated for 15 min with sulfo-NHS-biotin. After solubilization, biotinylated protein was recovered by streptavidin precipitation. The streptavidin fractions ( lanes labeled 2 ), representing the membrane proteins, and aliquots of the lysate before ( lanes labeled 1 ) and after ( lanes labeled 3 ) streptavidin precipitation were analyzed by immunoblotting using anti-NR1, anti-NR2A/B, anti-NR3A, and anti-calreticulin antibodies. An excess amount of protein was loaded in the lanes labeled 2 to ensure detection of any NR3A or calreticulin at the cell surface. The subunit combinations used for transfection are indicated above each blot, and the positions of molecular size markers in kilodaltons are shown on the left . A representative experiment is shown; n = 3. B, C , Surface localization of GFP-tagged NR3A. B, Left , Schematic drawing of expected transmembrane ( TM ) topology of NR3A-GFP is shown. Right , Protein immunoblots of HEK293T cells transfected with NR3A or NR3AGFP and probed with anti-NR3A antibody show an increase in NR3A molecular weight that corresponds to the molecular mass of GFP (27 kDa). No lower molecular weight bands were observed. C , Cells transfected with GFP-tagged NR3A alone or in combination with the other NMDA receptor subunits were immunostained in nonpermeabilizing (NP) conditions with anti-GFP antibody followed by a Texas Red-conjugated secondary antibody and imaged with filters for GFP and Texas Red. All four panels show raw superimposed confocal images combining NP anti-GFP antibody staining ( red ) and native GFP fluorescence from NR3A-GFP ( green ). Yellow corresponds to the overlap of GFP immunostaining and GFP fluorescence and reflects NR3A-GFP expressed at the cell surface. Because the intensity of red immunostaining was brighter than was green GFP fluorescence, regions of overlapping can appear red-yellow . When expressed alone, NR3A-GFP exhibits a perinuclear and reticular fluorescence pattern, and no surface staining is observed. Cotransfection of NR1-1a/NR2A leads to the appearance of patches of fluorescence at the plasma membrane. Scale bar, 10 μm.
    Figure Legend Snippet: NR3A is present at the cell surface only when coexpressed with the NR1-1a subunit. A , HEK293T cells were transfected with different combinations of NMDA receptor subunits and incubated for 15 min with sulfo-NHS-biotin. After solubilization, biotinylated protein was recovered by streptavidin precipitation. The streptavidin fractions ( lanes labeled 2 ), representing the membrane proteins, and aliquots of the lysate before ( lanes labeled 1 ) and after ( lanes labeled 3 ) streptavidin precipitation were analyzed by immunoblotting using anti-NR1, anti-NR2A/B, anti-NR3A, and anti-calreticulin antibodies. An excess amount of protein was loaded in the lanes labeled 2 to ensure detection of any NR3A or calreticulin at the cell surface. The subunit combinations used for transfection are indicated above each blot, and the positions of molecular size markers in kilodaltons are shown on the left . A representative experiment is shown; n = 3. B, C , Surface localization of GFP-tagged NR3A. B, Left , Schematic drawing of expected transmembrane ( TM ) topology of NR3A-GFP is shown. Right , Protein immunoblots of HEK293T cells transfected with NR3A or NR3AGFP and probed with anti-NR3A antibody show an increase in NR3A molecular weight that corresponds to the molecular mass of GFP (27 kDa). No lower molecular weight bands were observed. C , Cells transfected with GFP-tagged NR3A alone or in combination with the other NMDA receptor subunits were immunostained in nonpermeabilizing (NP) conditions with anti-GFP antibody followed by a Texas Red-conjugated secondary antibody and imaged with filters for GFP and Texas Red. All four panels show raw superimposed confocal images combining NP anti-GFP antibody staining ( red ) and native GFP fluorescence from NR3A-GFP ( green ). Yellow corresponds to the overlap of GFP immunostaining and GFP fluorescence and reflects NR3A-GFP expressed at the cell surface. Because the intensity of red immunostaining was brighter than was green GFP fluorescence, regions of overlapping can appear red-yellow . When expressed alone, NR3A-GFP exhibits a perinuclear and reticular fluorescence pattern, and no surface staining is observed. Cotransfection of NR1-1a/NR2A leads to the appearance of patches of fluorescence at the plasma membrane. Scale bar, 10 μm.

    Techniques Used: Transfection, Incubation, Labeling, Western Blot, Molecular Weight, Staining, Fluorescence, Immunostaining, Cotransfection

    54) Product Images from "The Mycobacterium tuberculosis Secreted Protein Rv0203 Transfers Heme to Membrane Proteins MmpL3 and MmpL11"

    Article Title: The Mycobacterium tuberculosis Secreted Protein Rv0203 Transfers Heme to Membrane Proteins MmpL3 and MmpL11

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.453076

    Heme transfer experiments. A, SDS-PAGE of the pulldown fractions for transfer analyses between Rv0203 and MmpL3-E1 and Rv0203 and MmpL11-E1. Lane 1, streptavidin beads after reaction with MmpL3-E1; lane 2, MmpL3-E1 flow-through; lane 3, streptavidin beads
    Figure Legend Snippet: Heme transfer experiments. A, SDS-PAGE of the pulldown fractions for transfer analyses between Rv0203 and MmpL3-E1 and Rv0203 and MmpL11-E1. Lane 1, streptavidin beads after reaction with MmpL3-E1; lane 2, MmpL3-E1 flow-through; lane 3, streptavidin beads

    Techniques Used: SDS Page, Flow Cytometry

    55) Product Images from "Proteomic examination of Leishmania chagasi plasma membrane proteins: contrast between avirulent and virulent (metacyclic) parasite forms"

    Article Title: Proteomic examination of Leishmania chagasi plasma membrane proteins: contrast between avirulent and virulent (metacyclic) parasite forms

    Journal: Proteomics. Clinical applications

    doi: 10.1002/prca.200900050

    Percentage coverage (% amino acid) ( A ) and number of predicted transmembrane domains (TMD) and GPI anchors ( B ) of the 447 proteins detected using surface biotinylation-streptavidin affinity purification and LC-MS/MS.
    Figure Legend Snippet: Percentage coverage (% amino acid) ( A ) and number of predicted transmembrane domains (TMD) and GPI anchors ( B ) of the 447 proteins detected using surface biotinylation-streptavidin affinity purification and LC-MS/MS.

    Techniques Used: Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Identification of plasma membrane proteins by biotin-avidin affinity purification and LC-MS/MS. A . A flow chart of surface biotinylation and streptavidin isolation of plasma membrane proteins (top panel), and western blots showing the abundances of the major surface proteases (MSP) and cleanness of the preparation (lane 4) without detectable contaminants of cytoskeletal α-tubulin (α-T), cytosolic protein 36 (P36) and the luminal protein BiP of the endoplasmic reticulum. B . Numbers of protein identified (vertical bars) and the average molecular sizes in kDa (solid line) from each gel slice by LC-MS/MS. The 40 gel slices were generated from a silver-stained 5–15% gradient SDS-PAGE gel strip loaded with the plasma-membrane proteins (Same as lane 4 in A ) of 2 × 10 9 cell equivalence.
    Figure Legend Snippet: Identification of plasma membrane proteins by biotin-avidin affinity purification and LC-MS/MS. A . A flow chart of surface biotinylation and streptavidin isolation of plasma membrane proteins (top panel), and western blots showing the abundances of the major surface proteases (MSP) and cleanness of the preparation (lane 4) without detectable contaminants of cytoskeletal α-tubulin (α-T), cytosolic protein 36 (P36) and the luminal protein BiP of the endoplasmic reticulum. B . Numbers of protein identified (vertical bars) and the average molecular sizes in kDa (solid line) from each gel slice by LC-MS/MS. The 40 gel slices were generated from a silver-stained 5–15% gradient SDS-PAGE gel strip loaded with the plasma-membrane proteins (Same as lane 4 in A ) of 2 × 10 9 cell equivalence.

    Techniques Used: Avidin-Biotin Assay, Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Flow Cytometry, Isolation, Western Blot, Generated, Staining, SDS Page, Stripping Membranes

    56) Product Images from "Myosin IIA associates with NK cell lytic granules to enable their interaction with F-actin and function at the immunological synapse 1"

    Article Title: Myosin IIA associates with NK cell lytic granules to enable their interaction with F-actin and function at the immunological synapse 1

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.0804337

    Functional association of lytic granules with F-actin and effects of ATP and myosin IIA 1933x. Isolated lytic granules were added to flow chambers containing biotinylated BSA/streptavidin (BSA/SA) without actin, with actin and physiologic salt buffer,
    Figure Legend Snippet: Functional association of lytic granules with F-actin and effects of ATP and myosin IIA 1933x. Isolated lytic granules were added to flow chambers containing biotinylated BSA/streptavidin (BSA/SA) without actin, with actin and physiologic salt buffer,

    Techniques Used: Functional Assay, Isolation, Flow Cytometry

    57) Product Images from "H3K27me1 is essential for MMP-9-dependent H3N-terminal tail proteolysis during osteoclastogenesis"

    Article Title: H3K27me1 is essential for MMP-9-dependent H3N-terminal tail proteolysis during osteoclastogenesis

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0193-1

    MMP-9 binding to H3K27me1 nucleosomes. a Schematic depiction of the domain structure of MMP-9. b Peptide pull-down assays with biotinylated H3 1–21 and 21–44 peptides and recombinant His-MMP-9 were analyzed by Western blotting with anti-His antibody. H3 peptides were unmodified, K18ac or K27me1 as indicated. Lane 1 represents 10% of the input MMP-9. c Nucleosomes were reconstituted on a 207-bp 601 nucleosome positioning sequence using unmodified or H3K27me1 histone octamers and immobilized on streptavidin beads. His-MMP-9 was incubated with immobilized nucleosomes, and its binding to nucleosomes was analyzed by Western blotting with anti-His antibody. Lane 1 contains 10% of the input MMP-9. d H3K27me1 nucleosomes were incubated with immobilized MMP-9 N-terminal (amino acids 112–447) and C-terminal (amino acids 448–730) domains. After extensive washing, the binding of H3K27me1 nucleosomes to MMP-9 domains was determined by Western blotting with anti-H3 antibody. Input corresponds to 10% of H3K27me1 nucleosomes used in the binding reactions. e After incubation with H3K27me1 nucleosomes, the binding of MMP-9N-terminal subregions to nucleosomes was determined by Western blotting with anti-His antibody. Input lanes 1–3 represent 10% of MMP-9 fragments used in the binding reactions. f OCP-induced cells were transfected with FLAG-H3 wild type (WT) or K27R mutant (K27R), and mononucleosomes were prepared by micrococcal nuclease digestion as summarized in Figure S3. Mononucleosomes containing ectopic H3 were immunoprecipitated from total mononucleosomes with FLAG antibody and analyzed by Western blotting with anti-MMP-9 antibody. g ]. Simulations were run with non-methylated H3. For context, H3K27 is shown monomethylated. h Nucleosome binding assays were conducted as in e , except that His-MMP-9 amino acids 384–447 carrying E402A mutation were used
    Figure Legend Snippet: MMP-9 binding to H3K27me1 nucleosomes. a Schematic depiction of the domain structure of MMP-9. b Peptide pull-down assays with biotinylated H3 1–21 and 21–44 peptides and recombinant His-MMP-9 were analyzed by Western blotting with anti-His antibody. H3 peptides were unmodified, K18ac or K27me1 as indicated. Lane 1 represents 10% of the input MMP-9. c Nucleosomes were reconstituted on a 207-bp 601 nucleosome positioning sequence using unmodified or H3K27me1 histone octamers and immobilized on streptavidin beads. His-MMP-9 was incubated with immobilized nucleosomes, and its binding to nucleosomes was analyzed by Western blotting with anti-His antibody. Lane 1 contains 10% of the input MMP-9. d H3K27me1 nucleosomes were incubated with immobilized MMP-9 N-terminal (amino acids 112–447) and C-terminal (amino acids 448–730) domains. After extensive washing, the binding of H3K27me1 nucleosomes to MMP-9 domains was determined by Western blotting with anti-H3 antibody. Input corresponds to 10% of H3K27me1 nucleosomes used in the binding reactions. e After incubation with H3K27me1 nucleosomes, the binding of MMP-9N-terminal subregions to nucleosomes was determined by Western blotting with anti-His antibody. Input lanes 1–3 represent 10% of MMP-9 fragments used in the binding reactions. f OCP-induced cells were transfected with FLAG-H3 wild type (WT) or K27R mutant (K27R), and mononucleosomes were prepared by micrococcal nuclease digestion as summarized in Figure S3. Mononucleosomes containing ectopic H3 were immunoprecipitated from total mononucleosomes with FLAG antibody and analyzed by Western blotting with anti-MMP-9 antibody. g ]. Simulations were run with non-methylated H3. For context, H3K27 is shown monomethylated. h Nucleosome binding assays were conducted as in e , except that His-MMP-9 amino acids 384–447 carrying E402A mutation were used

    Techniques Used: Binding Assay, Recombinant, Western Blot, Sequencing, Incubation, Transfection, Mutagenesis, Immunoprecipitation, Methylation

    58) Product Images from "Ret finger protein-like 3 promotes tumor cell growth by activating telomerase reverse transcriptase expression in human lung cancer cells"

    Article Title: Ret finger protein-like 3 promotes tumor cell growth by activating telomerase reverse transcriptase expression in human lung cancer cells

    Journal: Oncotarget

    doi:

    Pulldown and identification of tumor-specific hTERT promoter binding proteins (A) The potential hTERT promoter-binding proteins were pulled down, separated by the SDS-PAGE, and visualized by silver staining. A representative SDS-PAGE image is shown, and the arrow indicates the candidate hTERT promoter-binding protein. (B) Chromatin immunoprecipitation assays were carried out using the hTERT promoter from normal lung cells and lung cancer cells. PCR products of hTERT promoter (−378 to +60) were separated on 1% agarose gels. The IgG was used as a negative control. (C) Chromatin immunoprecipitation assays were done using antibody against AP-2. The PCR products of hTERT promoter (−378 to +60) were separated on 1% agarose gels. The streptavidin-agarose pulldown with hTERT promoter (−378 to +60) as probes was done. AP-2 was tested in the pulled down protein complex by immunoblot using antibody against AP-2. (D) The nuclear extracts of human lung normal and cancer cells were prepared for immunoprecipitation using an antibody against RFPL3 and then evaluated by immunoblot using antibody against AP-2. (E) Human lung cancer H1299 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of RFPL3 with AP-2 were examined by confocal microscopy analysis with a confocal microscope. Densitometric analysis was used to analyze quantitatively the binding of RFPL3 on hTERT promoter.
    Figure Legend Snippet: Pulldown and identification of tumor-specific hTERT promoter binding proteins (A) The potential hTERT promoter-binding proteins were pulled down, separated by the SDS-PAGE, and visualized by silver staining. A representative SDS-PAGE image is shown, and the arrow indicates the candidate hTERT promoter-binding protein. (B) Chromatin immunoprecipitation assays were carried out using the hTERT promoter from normal lung cells and lung cancer cells. PCR products of hTERT promoter (−378 to +60) were separated on 1% agarose gels. The IgG was used as a negative control. (C) Chromatin immunoprecipitation assays were done using antibody against AP-2. The PCR products of hTERT promoter (−378 to +60) were separated on 1% agarose gels. The streptavidin-agarose pulldown with hTERT promoter (−378 to +60) as probes was done. AP-2 was tested in the pulled down protein complex by immunoblot using antibody against AP-2. (D) The nuclear extracts of human lung normal and cancer cells were prepared for immunoprecipitation using an antibody against RFPL3 and then evaluated by immunoblot using antibody against AP-2. (E) Human lung cancer H1299 cells grown on chamber slides were cultivated for 24 h, and the subcellular localization and the colocalization of RFPL3 with AP-2 were examined by confocal microscopy analysis with a confocal microscope. Densitometric analysis was used to analyze quantitatively the binding of RFPL3 on hTERT promoter.

    Techniques Used: Binding Assay, SDS Page, Silver Staining, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Negative Control, Immunoprecipitation, Confocal Microscopy, Microscopy

    59) Product Images from "Binding of the 5?-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription"

    Article Title: Binding of the 5?-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks165

    Defining the protein interaction sequence. (A) A schematic diagram of the RNA probes used to define the interaction region. Numbers denote nucleic acid residue position and roman numerals denote stem–loop number. The boundary of the leader sequence of IBV (nt. 1–64) is marked by the box on the full-length 5′-UTR. (B) I nteraction of various deletion constructs of IBV 5′-UTR with MADP1. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with RNA probes spanning different regions of IBV 5′-UTR. The RNA–protein complexes were purified with streptavidin beads, resolved by SDS–PAGE and probed with antibody to Flag tag for the presence of Flag-tagged MADP1 protein. (C) Diagram showing the two mutants of 5′-UTRΔ2 containing either two point mutations which disrupt stem–loop I (5′-UTRΔ2M1) or a mutant restoring stem–loop I in 5′-UTRΔ2M1 (5′-UTRΔ2M2). (D) The essential role of a stem–loop I in the interaction between MADP1 and IBV 5′-UTR. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with 5′-UTRΔ2M1 and 5′-UTRΔ2M2, respectively. The RNA–protein complexes were purified with streptavidin beads, resolved by SDS–PAGE and probed with antibody to Flag tag for the presence of Flag-tagged MADP1 protein.
    Figure Legend Snippet: Defining the protein interaction sequence. (A) A schematic diagram of the RNA probes used to define the interaction region. Numbers denote nucleic acid residue position and roman numerals denote stem–loop number. The boundary of the leader sequence of IBV (nt. 1–64) is marked by the box on the full-length 5′-UTR. (B) I nteraction of various deletion constructs of IBV 5′-UTR with MADP1. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with RNA probes spanning different regions of IBV 5′-UTR. The RNA–protein complexes were purified with streptavidin beads, resolved by SDS–PAGE and probed with antibody to Flag tag for the presence of Flag-tagged MADP1 protein. (C) Diagram showing the two mutants of 5′-UTRΔ2 containing either two point mutations which disrupt stem–loop I (5′-UTRΔ2M1) or a mutant restoring stem–loop I in 5′-UTRΔ2M1 (5′-UTRΔ2M2). (D) The essential role of a stem–loop I in the interaction between MADP1 and IBV 5′-UTR. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with 5′-UTRΔ2M1 and 5′-UTRΔ2M2, respectively. The RNA–protein complexes were purified with streptavidin beads, resolved by SDS–PAGE and probed with antibody to Flag tag for the presence of Flag-tagged MADP1 protein.

    Techniques Used: Sequencing, Construct, Expressing, Purification, SDS Page, FLAG-tag, Mutagenesis

    Comparison of the 5′ UTR of IBV, SARS-CoV and HCoV-OC43. (A) Interaction of the 5′ UTR from IBV, SARS-CoV and HCoV-OC43 with MADP1. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with biotinylated RNA probes of full-length 5′ UTRs of the three coronaviruses. The RNA–protein complexes were purified with streptavidin beads, resolved by SDS–PAGE and probed with antibody to Flag tag for the presence of Flag-tagged MADP1 protein. (B) The predicted secondary structures of stem–loop I from IBV, SARS-CoV and HCoV-OC43.
    Figure Legend Snippet: Comparison of the 5′ UTR of IBV, SARS-CoV and HCoV-OC43. (A) Interaction of the 5′ UTR from IBV, SARS-CoV and HCoV-OC43 with MADP1. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with biotinylated RNA probes of full-length 5′ UTRs of the three coronaviruses. The RNA–protein complexes were purified with streptavidin beads, resolved by SDS–PAGE and probed with antibody to Flag tag for the presence of Flag-tagged MADP1 protein. (B) The predicted secondary structures of stem–loop I from IBV, SARS-CoV and HCoV-OC43.

    Techniques Used: Expressing, Purification, SDS Page, FLAG-tag

    MADP1 interacts specifically with IBV 5′-UTR. (A) Interaction of MADP1 with SARS-CoV and IBV 5′-UTR in a biotin-RNA pull-down assay. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with 0.1 µM of biotinylated IBV and SARS-CoV 5′-UTR, respectively, followed by addition of streptavidin agarose beads. Unbound complexes to the beads were subsequently removed by washing and complexes that remained bound to the beads were eluted with gel loading buffer. All fractions and elute were resolved by SDS–PAGE and probed with antibody to Flag tag. (B) Competition assay for the specificity of interaction between MADP1 and IBV 5′-UTR. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 was added to mixtures of 0.1 µM biotinylated IBV 5′-UTR RNA and varying concentrations of unlabeled IBV 5′-UTR or EGFP RNA. Streptavidin agarose beads were added and treated under conditions identical to (A). Total cell lysates prepared from cells over-expressing Flag-tagged IBV N protein were added to mixtures of a fixed concentration of biotinylated IBV 3′-UTR RNA and unlabeled IBV 3′-UTR or EGFP RNA and subjected to the same treatment.
    Figure Legend Snippet: MADP1 interacts specifically with IBV 5′-UTR. (A) Interaction of MADP1 with SARS-CoV and IBV 5′-UTR in a biotin-RNA pull-down assay. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with 0.1 µM of biotinylated IBV and SARS-CoV 5′-UTR, respectively, followed by addition of streptavidin agarose beads. Unbound complexes to the beads were subsequently removed by washing and complexes that remained bound to the beads were eluted with gel loading buffer. All fractions and elute were resolved by SDS–PAGE and probed with antibody to Flag tag. (B) Competition assay for the specificity of interaction between MADP1 and IBV 5′-UTR. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 was added to mixtures of 0.1 µM biotinylated IBV 5′-UTR RNA and varying concentrations of unlabeled IBV 5′-UTR or EGFP RNA. Streptavidin agarose beads were added and treated under conditions identical to (A). Total cell lysates prepared from cells over-expressing Flag-tagged IBV N protein were added to mixtures of a fixed concentration of biotinylated IBV 3′-UTR RNA and unlabeled IBV 3′-UTR or EGFP RNA and subjected to the same treatment.

    Techniques Used: Pull Down Assay, Expressing, SDS Page, FLAG-tag, Competitive Binding Assay, Concentration Assay

    Defining the RNA-binding domain. (A) A schematic diagram of constructs of MADP1 and its truncation mutants. Numbers denote amino acid residue positions. Conserved domains RNA Recognition Motif (RRM) and Universal Minicircle Sequence Binding Protein (UMSBP) were indicated as black and white blocks, respectively. (B) Interaction of deletion mutants of MADP1 with IBV 5′-UTR. Cell lysates prepared from H1299 cells over-expressing Flag-tagged wild-type MADP1 or its truncation mutants were used for biotin-RNA pull-down assay using the full-length IBV 5′-UTR. Both the crude lysates (labeled C) and protein bound on the streptavidin beads (labeled E) were resolved by SDS–PAGE and detected by Western blot with anti-Flag antibody. EGFP over-expressed cell lysate was included as a negative control. (C) Interaction of three MADP1 mutant constructs, Y13A, V53F55A and YVF, with IBV 5′-UTR. The three full-length MADP1 constructs with amino acid mutations at the predicted RNA-binding sites were transfected into H1299 cells and used in a biotin-RNA pull-down assay with the full-length IBV 5′-UTR.
    Figure Legend Snippet: Defining the RNA-binding domain. (A) A schematic diagram of constructs of MADP1 and its truncation mutants. Numbers denote amino acid residue positions. Conserved domains RNA Recognition Motif (RRM) and Universal Minicircle Sequence Binding Protein (UMSBP) were indicated as black and white blocks, respectively. (B) Interaction of deletion mutants of MADP1 with IBV 5′-UTR. Cell lysates prepared from H1299 cells over-expressing Flag-tagged wild-type MADP1 or its truncation mutants were used for biotin-RNA pull-down assay using the full-length IBV 5′-UTR. Both the crude lysates (labeled C) and protein bound on the streptavidin beads (labeled E) were resolved by SDS–PAGE and detected by Western blot with anti-Flag antibody. EGFP over-expressed cell lysate was included as a negative control. (C) Interaction of three MADP1 mutant constructs, Y13A, V53F55A and YVF, with IBV 5′-UTR. The three full-length MADP1 constructs with amino acid mutations at the predicted RNA-binding sites were transfected into H1299 cells and used in a biotin-RNA pull-down assay with the full-length IBV 5′-UTR.

    Techniques Used: RNA Binding Assay, Construct, Sequencing, Binding Assay, Expressing, Pull Down Assay, Labeling, SDS Page, Western Blot, Negative Control, Mutagenesis, Transfection

    60) Product Images from "Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions"

    Article Title: Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

    Journal: Journal of Virology

    doi: 10.1128/JVI.01258-13

    A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins
    Figure Legend Snippet: A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins

    Techniques Used: Western Blot, Infection

    Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The
    Figure Legend Snippet: Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The

    Techniques Used: Expressing, Construct, Recombinant, Binding Assay

    61) Product Images from "SUMO1 negatively regulates BRCA1-mediated transcription, via modulation of promoter occupancy"

    Article Title: SUMO1 negatively regulates BRCA1-mediated transcription, via modulation of promoter occupancy

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm969

    SUMO1 inhibits binding of BRCA1 to the Gadd45α promoter. ( A ) BRCA1-Gadd45α promoter-binding assay was performed with nuclear extract from cells transiently expressing HA-BRCA1, FLAG-BARD1 and/or SUMO1. Biotin-labeled Gadd45α promoter (−107 to −57) DNA was incubated with nuclear extract at room temperature in binding buffer. Following incubation, BRCA1 protein bound to biotin-labeled Gadd45α promoter DNA was isolated with streptavidin-agarose. The BRCA1–DNA–streptavidin–agarose complex was loaded onto a SDS gel. Proteins bound to DNA were detected by immunoblotting with anti-HA (for HA-BRCA1), anti-FLAG (for FLAG-BARD1) or anti-SUMO1 (for SUMO1) antibodies. The level of exogenous HA-BRCA1, FLAG-BARD1 and RFP-SUMO1 proteins were evaluated by immunoblotting with anti-HA, anti-FLAG and anti-SUMO1 antibodies, respectively. The representative figure was shown from seven separate experiments. ( B ) BRCA1-Gadd45α promoter-binding assay was performed as described in (A) with nuclear extract from cells transiently expressing either wild-type SUMO1 or its mutant derivative SUMO1ΔGG alone. Endogenous BRCA1 and BARD1 proteins or exogenous RFP-SUMO1 and RFP- SUMO1ΔGG bound to DNA were detected by immunoblotting with anti-BRCA1, anti-BARD1 and anti-SUMO1 antibodies, respectively. The representative figure was shown from three separate experiments.
    Figure Legend Snippet: SUMO1 inhibits binding of BRCA1 to the Gadd45α promoter. ( A ) BRCA1-Gadd45α promoter-binding assay was performed with nuclear extract from cells transiently expressing HA-BRCA1, FLAG-BARD1 and/or SUMO1. Biotin-labeled Gadd45α promoter (−107 to −57) DNA was incubated with nuclear extract at room temperature in binding buffer. Following incubation, BRCA1 protein bound to biotin-labeled Gadd45α promoter DNA was isolated with streptavidin-agarose. The BRCA1–DNA–streptavidin–agarose complex was loaded onto a SDS gel. Proteins bound to DNA were detected by immunoblotting with anti-HA (for HA-BRCA1), anti-FLAG (for FLAG-BARD1) or anti-SUMO1 (for SUMO1) antibodies. The level of exogenous HA-BRCA1, FLAG-BARD1 and RFP-SUMO1 proteins were evaluated by immunoblotting with anti-HA, anti-FLAG and anti-SUMO1 antibodies, respectively. The representative figure was shown from seven separate experiments. ( B ) BRCA1-Gadd45α promoter-binding assay was performed as described in (A) with nuclear extract from cells transiently expressing either wild-type SUMO1 or its mutant derivative SUMO1ΔGG alone. Endogenous BRCA1 and BARD1 proteins or exogenous RFP-SUMO1 and RFP- SUMO1ΔGG bound to DNA were detected by immunoblotting with anti-BRCA1, anti-BARD1 and anti-SUMO1 antibodies, respectively. The representative figure was shown from three separate experiments.

    Techniques Used: Binding Assay, Expressing, Labeling, Incubation, Isolation, SDS-Gel, Mutagenesis

    62) Product Images from "Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress"

    Article Title: Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress

    Journal: Diseases

    doi: 10.3390/diseases6030064

    Analysis of viral PPxY-host WW-domain interactions between BAG3 and LFV-Z by peptide pull-down assays. ( A ) Flow chart of the peptide pull-down assay using LFV-Z peptides and cell lysates expressing BAG3-WT; ( B ) Schematic diagram of BAG3-WT, BAG3-ΔN, and BAG3-ΔC mutants with the various domains highlighted in color and amino acid positions indicated; ( C ) Western blot of peptide pull-down assay using streptavidin agarose beads conjugated with either the LFV-Z WT or LFV-Z PY mutant peptide. BAG3 proteins were detected using anti-c-myc antibody (top blot). Expression controls for BAG3 and actin are shown in the bottom blot. These results are from 1 of 2 independent experiments.
    Figure Legend Snippet: Analysis of viral PPxY-host WW-domain interactions between BAG3 and LFV-Z by peptide pull-down assays. ( A ) Flow chart of the peptide pull-down assay using LFV-Z peptides and cell lysates expressing BAG3-WT; ( B ) Schematic diagram of BAG3-WT, BAG3-ΔN, and BAG3-ΔC mutants with the various domains highlighted in color and amino acid positions indicated; ( C ) Western blot of peptide pull-down assay using streptavidin agarose beads conjugated with either the LFV-Z WT or LFV-Z PY mutant peptide. BAG3 proteins were detected using anti-c-myc antibody (top blot). Expression controls for BAG3 and actin are shown in the bottom blot. These results are from 1 of 2 independent experiments.

    Techniques Used: Flow Cytometry, Pull Down Assay, Expressing, Western Blot, Mutagenesis

    63) Product Images from "Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation, although Furin Cleavage Alone Generates Inactive Proteinase *"

    Article Title: Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation, although Furin Cleavage Alone Generates Inactive Proteinase *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.135442

    Cell surface expression of the prodomain mutants in HT1080 cells. A , cell surface expression of the prodomain mutants in HT1080 cells. Biotin-labeled cell surface proteins were immunoprecipitated ( IP ) using streptavidin-agarose beads. The precipitates
    Figure Legend Snippet: Cell surface expression of the prodomain mutants in HT1080 cells. A , cell surface expression of the prodomain mutants in HT1080 cells. Biotin-labeled cell surface proteins were immunoprecipitated ( IP ) using streptavidin-agarose beads. The precipitates

    Techniques Used: Expressing, Labeling, Immunoprecipitation

    64) Product Images from "Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry"

    Article Title: Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

    Journal: Journal of Virology

    doi: 10.1128/JVI.01015-17

    Transmission electron microscopy of EdU-labeled cells. (A) A549 cells were infected with VACV and at 3.5 h were incubated with EdU for 30 min. The cells were fixed and permeabilized with 0.1% Triton X-100 and reacted with biotin-azide, followed by streptavidin–6-nm gold. (B) Same as panel A, except that biotin-azide was omitted as a control. (C) A549 cells were infected and labeled with EdU as for panel A. The cells were then permeabilized with 0.05% saponin and reacted with biotin-azide. The cells were cryosectioned and incubated with streptavidin–10-nm gold. (D) Same as panel C, except that biotin-azide was omitted as a control. Nu, nucleus; Go, Golgi apparatus; Mi, mitochondria.
    Figure Legend Snippet: Transmission electron microscopy of EdU-labeled cells. (A) A549 cells were infected with VACV and at 3.5 h were incubated with EdU for 30 min. The cells were fixed and permeabilized with 0.1% Triton X-100 and reacted with biotin-azide, followed by streptavidin–6-nm gold. (B) Same as panel A, except that biotin-azide was omitted as a control. (C) A549 cells were infected and labeled with EdU as for panel A. The cells were then permeabilized with 0.05% saponin and reacted with biotin-azide. The cells were cryosectioned and incubated with streptavidin–10-nm gold. (D) Same as panel C, except that biotin-azide was omitted as a control. Nu, nucleus; Go, Golgi apparatus; Mi, mitochondria.

    Techniques Used: Transmission Assay, Electron Microscopy, Labeling, Infection, Incubation

    65) Product Images from "The tuberous sclerosis complex subunit TBC1D7 is stabilized by Akt phosphorylation–mediated 14-3-3 binding"

    Article Title: The tuberous sclerosis complex subunit TBC1D7 is stabilized by Akt phosphorylation–mediated 14-3-3 binding

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003525

    PHLPP proteins and TBC1D7 are binding partners. A , 293T cells were transfected with either SBP or SBP-PHLPP1 expression plasmids. Lysates were subject to pulldown analysis using streptavidin beads. Affinity-purified complexes were resolved on SDS-PAGE,
    Figure Legend Snippet: PHLPP proteins and TBC1D7 are binding partners. A , 293T cells were transfected with either SBP or SBP-PHLPP1 expression plasmids. Lysates were subject to pulldown analysis using streptavidin beads. Affinity-purified complexes were resolved on SDS-PAGE,

    Techniques Used: Binding Assay, Transfection, Expressing, Affinity Purification, SDS Page

    Ser-124 phosphorylation stabilizes TBC1D7. A , 293T cells were transfected with either SBP vector or SBP-TBC1D7 WT, S124A, S124E expression plasmids. Lysates were subject to pulldown analysis with streptavidin beads. Affinity-purified complexes and input
    Figure Legend Snippet: Ser-124 phosphorylation stabilizes TBC1D7. A , 293T cells were transfected with either SBP vector or SBP-TBC1D7 WT, S124A, S124E expression plasmids. Lysates were subject to pulldown analysis with streptavidin beads. Affinity-purified complexes and input

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Affinity Purification

    66) Product Images from "Nitro-fatty acid inhibition of triple-negative breast cancer cell viability, migration, invasion, and tumor growth"

    Article Title: Nitro-fatty acid inhibition of triple-negative breast cancer cell viability, migration, invasion, and tumor growth

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.814368

    NO 2 -OA inhibits TNFα-induced I KKβ phosphorylation and IκBα degradation and covalently adducts IKKβ . MDA-MB-231 and MDA-MB-468 cells were used in all studies. A , representative immunoblot of IKKβ (Ser-180) phosphorylation, total IKKβ levels, and relative phosphorylated IKKβ levels. Then all phosphorylated IKKβ levels normalized to total IKKβ were quantified. B , representative immunoblot of IκBα protein levels is shown, and the relative total IκBα levels (normalized to total β-actin) are quantified in response to NO 2 -SA, NO 2 -OA, and the NF-κB inhibitor BAY11-7082. C , representative immunoblots of IκBα (Ser-32) phosphorylation and total IκBα are shown in response to NO 2 -SA, NO 2 -OA, and the NF-κB inhibitor BAY11-7082. D , NO 2 -OA alkylates TNBC IKKβ protein. Biotinylated NO 2 -OA, NO 2 -SA, and OA and adducted proteins were affinity-purified by streptavidin-agarose beads from cell lysates. Pulled-down IKKβ protein was then detected by immunoblotting. IKKβ and control β-actin immunoblots from the same input lysates used for affinity purification are shown below the panel . *, p
    Figure Legend Snippet: NO 2 -OA inhibits TNFα-induced I KKβ phosphorylation and IκBα degradation and covalently adducts IKKβ . MDA-MB-231 and MDA-MB-468 cells were used in all studies. A , representative immunoblot of IKKβ (Ser-180) phosphorylation, total IKKβ levels, and relative phosphorylated IKKβ levels. Then all phosphorylated IKKβ levels normalized to total IKKβ were quantified. B , representative immunoblot of IκBα protein levels is shown, and the relative total IκBα levels (normalized to total β-actin) are quantified in response to NO 2 -SA, NO 2 -OA, and the NF-κB inhibitor BAY11-7082. C , representative immunoblots of IκBα (Ser-32) phosphorylation and total IκBα are shown in response to NO 2 -SA, NO 2 -OA, and the NF-κB inhibitor BAY11-7082. D , NO 2 -OA alkylates TNBC IKKβ protein. Biotinylated NO 2 -OA, NO 2 -SA, and OA and adducted proteins were affinity-purified by streptavidin-agarose beads from cell lysates. Pulled-down IKKβ protein was then detected by immunoblotting. IKKβ and control β-actin immunoblots from the same input lysates used for affinity purification are shown below the panel . *, p

    Techniques Used: Multiple Displacement Amplification, Western Blot, Affinity Purification

    67) Product Images from "Three Proteins of the U7-Specific Sm Ring Function as the Molecular Ruler To Determine the Site of 3?-End Processing in Mammalian Histone Pre-mRNA ▿Three Proteins of the U7-Specific Sm Ring Function as the Molecular Ruler To Determine the Site of 3?-End Processing in Mammalian Histone Pre-mRNA ▿ †"

    Article Title: Three Proteins of the U7-Specific Sm Ring Function as the Molecular Ruler To Determine the Site of 3?-End Processing in Mammalian Histone Pre-mRNA ▿Three Proteins of the U7-Specific Sm Ring Function as the Molecular Ruler To Determine the Site of 3?-End Processing in Mammalian Histone Pre-mRNA ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00296-09

    Mapping the interaction sites in histone pre-mRNA. (A) Sequences of biotinylated RNA halves. The U7-binding site is underlined, and nucleotide substitutions within the purine core of the mutant DCP RNA are indicated with lowercase letters. (B) Western blot analysis of affinity-purified complexes assembled on 5′ biotinylated RNAs (12.5 pmol), as indicated at the top of each lane. Processing complexes were formed in the presence of 0.2% NP-40 by using RNA substrates shown in panel A and subsequently purified on streptavidin beads. The presence of individual proteins bound to each RNA was detected by specific antibodies. In the top panel, anti-mouse 3′hExo and anti-SLBP antibodies were mixed. Lane 1 contains 10% of the extract used to assemble processing complexes on each RNA species.
    Figure Legend Snippet: Mapping the interaction sites in histone pre-mRNA. (A) Sequences of biotinylated RNA halves. The U7-binding site is underlined, and nucleotide substitutions within the purine core of the mutant DCP RNA are indicated with lowercase letters. (B) Western blot analysis of affinity-purified complexes assembled on 5′ biotinylated RNAs (12.5 pmol), as indicated at the top of each lane. Processing complexes were formed in the presence of 0.2% NP-40 by using RNA substrates shown in panel A and subsequently purified on streptavidin beads. The presence of individual proteins bound to each RNA was detected by specific antibodies. In the top panel, anti-mouse 3′hExo and anti-SLBP antibodies were mixed. Lane 1 contains 10% of the extract used to assemble processing complexes on each RNA species.

    Techniques Used: Binding Assay, Mutagenesis, Western Blot, Affinity Purification, Purification

    Affinity purification of a stable processing complex assembled on histone pre-mRNA. (A) Sequence of the 62-nucleotide FL RNA containing all elements required for 3′-end processing. The cleavage site is indicated with a vertical arrow, and the HDE that base pairs with the U7 snRNA is underlined. The biotin tag is located at the 5′ end of the RNA. (B) In vitro 3′-end processing of a 5′-labeled 86-nucleotide substrate RNA (0.05 pmol/reaction) that is nearly identical with the RNA shown in panel A but lacks the biotin tag and contains additional nucleotides at the 5′ and 3′ ends. Processing was carried out with a mouse nuclear extract (NE) under control conditions (lane 2) or in the presence of the indicated concentrations of NP-40. Lane 1 contains only the input RNA. (C) Coomassie-stained SDS-polyacrylamide gel containing proteins of a mouse nuclear extract adsorbed on streptavidin beads in the presence of 125 pmol of the biotinylated FL RNA (lane 1) or in its absence (lane 2). Processing complexes were formed in the presence of 0.2% NP-40 and subsequently purified on streptavidin beads. The sample lacking the RNA was four times smaller than the RNA-containing sample. Protein identities determined by MS are shown to the left, and size markers are to the right. In addition to FL proteins, MS identified a number of their degradation products. (D) Coomassie-stained SDS-polyacrylamide gel containing proteins of a mouse nuclear extract that associated with the biotinylated FL RNA (125 pmol) in the absence (lane 1) or presence (lane 2) of the anti-mU7 oligonucleotide. Protein bands present only in lane 1 are indicated with asterisks. (E) A fraction of material shown in D was analyzed by Western blotting with anti-hnRNP Q/R (top) or mixed antibodies to 3′hExo and SLBP (bottom). “NS” indicates cross-reacting proteins.
    Figure Legend Snippet: Affinity purification of a stable processing complex assembled on histone pre-mRNA. (A) Sequence of the 62-nucleotide FL RNA containing all elements required for 3′-end processing. The cleavage site is indicated with a vertical arrow, and the HDE that base pairs with the U7 snRNA is underlined. The biotin tag is located at the 5′ end of the RNA. (B) In vitro 3′-end processing of a 5′-labeled 86-nucleotide substrate RNA (0.05 pmol/reaction) that is nearly identical with the RNA shown in panel A but lacks the biotin tag and contains additional nucleotides at the 5′ and 3′ ends. Processing was carried out with a mouse nuclear extract (NE) under control conditions (lane 2) or in the presence of the indicated concentrations of NP-40. Lane 1 contains only the input RNA. (C) Coomassie-stained SDS-polyacrylamide gel containing proteins of a mouse nuclear extract adsorbed on streptavidin beads in the presence of 125 pmol of the biotinylated FL RNA (lane 1) or in its absence (lane 2). Processing complexes were formed in the presence of 0.2% NP-40 and subsequently purified on streptavidin beads. The sample lacking the RNA was four times smaller than the RNA-containing sample. Protein identities determined by MS are shown to the left, and size markers are to the right. In addition to FL proteins, MS identified a number of their degradation products. (D) Coomassie-stained SDS-polyacrylamide gel containing proteins of a mouse nuclear extract that associated with the biotinylated FL RNA (125 pmol) in the absence (lane 1) or presence (lane 2) of the anti-mU7 oligonucleotide. Protein bands present only in lane 1 are indicated with asterisks. (E) A fraction of material shown in D was analyzed by Western blotting with anti-hnRNP Q/R (top) or mixed antibodies to 3′hExo and SLBP (bottom). “NS” indicates cross-reacting proteins.

    Techniques Used: Affinity Purification, Sequencing, In Vitro, Labeling, Staining, Purification, Mass Spectrometry, Western Blot

    68) Product Images from "Quantitative analysis of the human T cell palmitome"

    Article Title: Quantitative analysis of the human T cell palmitome

    Journal: Scientific Reports

    doi: 10.1038/srep11598

    Experimental workflow for the detection of palmitoylated proteins from primary human T cells. Pooled lysates from healthy human donors are initially divided into “Enriched” and “Control” samples. Disulfide bonds are reduced by tris(2-carboxyethyl)phosphine (TCEP), and free thiols are blocked by N-ethylmaleimide (NEM). Following cleavage of the palmitoyl thioester bond by hydroxylamine (HA) in the enriched sample, previously palmitoylated thiols are biotinylated using the sulfhydryl-reactive EZ-Link HPDP-biotin. Control samples omit HA cleavage, and remain unbiotinylated. Following enrichment via streptavidin-agarose beads and elution by beta-mercaptoethanol (2-ME), enriched and control samples are run in parallel lanes of an SDS-PAGE gel. These lanes are cut into equal-sized bands and subjected to tryptic digest in either heavy (H 2 18 O) or light (H 2 16 O) water, providing the isotopic label. After digestion, samples are mixed and measured by LC-MS/MS; evaluating the isotopic heavy/light intensity ratio gives rise to an enriched pool representing the population of palmitoylated proteins in the cell.
    Figure Legend Snippet: Experimental workflow for the detection of palmitoylated proteins from primary human T cells. Pooled lysates from healthy human donors are initially divided into “Enriched” and “Control” samples. Disulfide bonds are reduced by tris(2-carboxyethyl)phosphine (TCEP), and free thiols are blocked by N-ethylmaleimide (NEM). Following cleavage of the palmitoyl thioester bond by hydroxylamine (HA) in the enriched sample, previously palmitoylated thiols are biotinylated using the sulfhydryl-reactive EZ-Link HPDP-biotin. Control samples omit HA cleavage, and remain unbiotinylated. Following enrichment via streptavidin-agarose beads and elution by beta-mercaptoethanol (2-ME), enriched and control samples are run in parallel lanes of an SDS-PAGE gel. These lanes are cut into equal-sized bands and subjected to tryptic digest in either heavy (H 2 18 O) or light (H 2 16 O) water, providing the isotopic label. After digestion, samples are mixed and measured by LC-MS/MS; evaluating the isotopic heavy/light intensity ratio gives rise to an enriched pool representing the population of palmitoylated proteins in the cell.

    Techniques Used: SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    69) Product Images from "Correlated matrix-assisted laser desorption/ionization mass spectrometry and fluorescent imaging of photocleavable peptide-coded random bead-arrays"

    Article Title: Correlated matrix-assisted laser desorption/ionization mass spectrometry and fluorescent imaging of photocleavable peptide-coded random bead-arrays

    Journal: Rapid Communications in Mass Spectrometry

    doi: 10.1002/rcm.6754

    Synchronized MALDI-MSI and fluorescence imaging of 20-member Mass-Tag bead-array. (A) Color-coded MALDI-MSI image of a 5920 × 3640 µm 2 region of a 20-member photocleavable Mass-Tag bead-array (photocleavable biotin peptide Mass-Tags loaded onto streptavidin glass beads). Corresponding masses ( m/z ) for Mass-Tags from representative beads (white circles) are indicated. (B) Color-coded overlaid MALDI-MSI spectra are shown for representative beads in the array (the beads indicated by white circles in (A)). Observed monoisotopic masses are listed. The ‘x-Axis Expansion’ inset shows a zoomed view of two distinct Mass-Tags of similar size, indicating a high mass resolution which can discriminate Mass-Tags separated by approximately 4 m/z units as well as the natural isotopes for each, spaced by 1 m/z unit. (C) One Mass-Tagged bead species in the array was labeled with fluorophore (e.g. as in Fig. 3 (A)). The fluorescence image of this bead species (same region as in panel (A)) is shown (gray/white spots) synchronized with a MALDI-MSI image of its cognate Mass-Tag (blue). For simplicity, MALDI-MSI images for two other Mass-Tags on non-fluorescent bead species are also superimposed (yellow and green).
    Figure Legend Snippet: Synchronized MALDI-MSI and fluorescence imaging of 20-member Mass-Tag bead-array. (A) Color-coded MALDI-MSI image of a 5920 × 3640 µm 2 region of a 20-member photocleavable Mass-Tag bead-array (photocleavable biotin peptide Mass-Tags loaded onto streptavidin glass beads). Corresponding masses ( m/z ) for Mass-Tags from representative beads (white circles) are indicated. (B) Color-coded overlaid MALDI-MSI spectra are shown for representative beads in the array (the beads indicated by white circles in (A)). Observed monoisotopic masses are listed. The ‘x-Axis Expansion’ inset shows a zoomed view of two distinct Mass-Tags of similar size, indicating a high mass resolution which can discriminate Mass-Tags separated by approximately 4 m/z units as well as the natural isotopes for each, spaced by 1 m/z unit. (C) One Mass-Tagged bead species in the array was labeled with fluorophore (e.g. as in Fig. 3 (A)). The fluorescence image of this bead species (same region as in panel (A)) is shown (gray/white spots) synchronized with a MALDI-MSI image of its cognate Mass-Tag (blue). For simplicity, MALDI-MSI images for two other Mass-Tags on non-fluorescent bead species are also superimposed (yellow and green).

    Techniques Used: Fluorescence, Imaging, Labeling

    MALDI-MSI imaging of single beads carrying photocleavable peptide Mass-Tags. (A) Two species of 30 µm streptavidin glass beads were prepared, pooled and used to form a two-dimensionally ordered, random bead-array. ‘Bead 1’ carried a single photocleavable biotin peptide Mass-Tag (blue) and a fluorophore (magenta ‘F’). ‘Bead 2’ carried two different photocleavable biotin peptide Mass-Tags (red and green) but no fluorophore. (B) (‘MALDI’) Color-coded MALDI-MSI image of a 1360 by 800 µm 2 region of the bead-array. Co-localization of the red and green Mass-Tags on Bead 2 appears as yellow. (‘Fluor’) Fluorescence image of same region of the bead-array, showing Bead 1. (‘MALDI Fluor’) Synchronized MALDI-MSI and fluorescence images showing co-localization of the fluorescence marker on Bead 1 (magenta) with the expected Mass-Tag (blue). (C) Color-coded MALDI spectra are shown from the center pixel of representative beads (the beads indicated by white arrows in (B)). The blue spectrum is from Bead 1 and yellow from Bead 2. Observed monoisotopic masses of the Mass-Tags are labeled in the spectra (note that while the scaling of the spectra does not allow visual discrimination of the natural isotopes of each Mass-Tag, separated by 1 m/z , they are resolved; for example, see Fig. 4 (B) ‘x-Axis Expansion’ inset).
    Figure Legend Snippet: MALDI-MSI imaging of single beads carrying photocleavable peptide Mass-Tags. (A) Two species of 30 µm streptavidin glass beads were prepared, pooled and used to form a two-dimensionally ordered, random bead-array. ‘Bead 1’ carried a single photocleavable biotin peptide Mass-Tag (blue) and a fluorophore (magenta ‘F’). ‘Bead 2’ carried two different photocleavable biotin peptide Mass-Tags (red and green) but no fluorophore. (B) (‘MALDI’) Color-coded MALDI-MSI image of a 1360 by 800 µm 2 region of the bead-array. Co-localization of the red and green Mass-Tags on Bead 2 appears as yellow. (‘Fluor’) Fluorescence image of same region of the bead-array, showing Bead 1. (‘MALDI Fluor’) Synchronized MALDI-MSI and fluorescence images showing co-localization of the fluorescence marker on Bead 1 (magenta) with the expected Mass-Tag (blue). (C) Color-coded MALDI spectra are shown from the center pixel of representative beads (the beads indicated by white arrows in (B)). The blue spectrum is from Bead 1 and yellow from Bead 2. Observed monoisotopic masses of the Mass-Tags are labeled in the spectra (note that while the scaling of the spectra does not allow visual discrimination of the natural isotopes of each Mass-Tag, separated by 1 m/z , they are resolved; for example, see Fig. 4 (B) ‘x-Axis Expansion’ inset).

    Techniques Used: Imaging, Fluorescence, Marker, Labeling

    Bead configuration in Bead-GPS. (A) Mass-Tags, e.g. peptides, are end-labeled with photocleavable biotin using an NHS-activated reagent and then captured onto streptavidin-coated 30 µm glass or 34 µm agarose beads (glass beads depicted; green numbers indicate the sequence of steps). (B) A novel photocleavable primary amine-terminated linker (NHS-PC-tBOC Linker) is attached to 30 µm mono-sized TentaGel® beads, which provides through its primary amine group a substrate for combinatorial synthesis of photocleavable peptide or peptoid libraries. Alternatively, independently synthesized Mass-Tags, e.g. peptides, can be attached to the photocleavable primary amine-terminated linker on the beads. (A and B) Streptavidin (tetrameric) is attached to the bead surface by a non-cleavable biotin (indicated by ‘B’ in figure). In addition to binding the Mass-Tags in some cases (e.g. as in (A)), the streptavidin coating can also facilitate attachment of separate ‘Bait’ molecules such as whole proteins using either a non-cleavable biotin (indicated by ‘B’ in figure) or a streptavidin binding tag (not depicted). See Experimental section for more detail.
    Figure Legend Snippet: Bead configuration in Bead-GPS. (A) Mass-Tags, e.g. peptides, are end-labeled with photocleavable biotin using an NHS-activated reagent and then captured onto streptavidin-coated 30 µm glass or 34 µm agarose beads (glass beads depicted; green numbers indicate the sequence of steps). (B) A novel photocleavable primary amine-terminated linker (NHS-PC-tBOC Linker) is attached to 30 µm mono-sized TentaGel® beads, which provides through its primary amine group a substrate for combinatorial synthesis of photocleavable peptide or peptoid libraries. Alternatively, independently synthesized Mass-Tags, e.g. peptides, can be attached to the photocleavable primary amine-terminated linker on the beads. (A and B) Streptavidin (tetrameric) is attached to the bead surface by a non-cleavable biotin (indicated by ‘B’ in figure). In addition to binding the Mass-Tags in some cases (e.g. as in (A)), the streptavidin coating can also facilitate attachment of separate ‘Bait’ molecules such as whole proteins using either a non-cleavable biotin (indicated by ‘B’ in figure) or a streptavidin binding tag (not depicted). See Experimental section for more detail.

    Techniques Used: Labeling, Sequencing, Synthesized, Binding Assay

    70) Product Images from "The Th17 immune response is controlled by the Rel-ROR?-ROR?T transcriptional axis"

    Article Title: The Th17 immune response is controlled by the Rel-ROR?-ROR?T transcriptional axis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20110462

    c-Rel binds to and activates the Rorgt promoter through two Rel sites. (A) WT and Rel site-mutated Rorgt promoter constructs and the empty vector were analyzed in a luciferase reporter assay with c-Rel co-transfection. The “X” indicates the mutated (Mu) Rel site. Data are representative of three independent experiments. (B) Nuclear extracts were prepared from EL4 cells after stimulation for 6 h with PMA and ionomycin. Biotinylated Rorgt Rel oligonucleotides or their mutants were absorbed by streptavidin-agarose beads, and then added to the nuclear extracts. The amount of c-Rel proteins in the precipitates were assessed by immunoblotting with anti–c-Rel. (C) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation condition as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel or control IgG. Data are representative of two independent experiments. (D) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation conditions, as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel. Data are representative of two independent experiments.
    Figure Legend Snippet: c-Rel binds to and activates the Rorgt promoter through two Rel sites. (A) WT and Rel site-mutated Rorgt promoter constructs and the empty vector were analyzed in a luciferase reporter assay with c-Rel co-transfection. The “X” indicates the mutated (Mu) Rel site. Data are representative of three independent experiments. (B) Nuclear extracts were prepared from EL4 cells after stimulation for 6 h with PMA and ionomycin. Biotinylated Rorgt Rel oligonucleotides or their mutants were absorbed by streptavidin-agarose beads, and then added to the nuclear extracts. The amount of c-Rel proteins in the precipitates were assessed by immunoblotting with anti–c-Rel. (C) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation condition as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel or control IgG. Data are representative of two independent experiments. (D) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation conditions, as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel. Data are representative of two independent experiments.

    Techniques Used: Construct, Plasmid Preparation, Luciferase, Reporter Assay, Cotransfection, Purification, Mouse Assay, Cell Culture, Chromatin Immunoprecipitation

    c-Rel binds to and activates the Rorg promoter through two specific Rel sites. (A) EL4 cells were transiently transfected with murine Rorg promoter luciferase construct together with an expression vector for full-length c-Rel, p65, p50, or RelB, or the empty vector as indicated. After 24 h, cells were treated with PMA and ionomycin for 5 h, and the luciferase activities measured. The promoter activity is presented as fold increase over cells transfected with empty vector. To normalize the transfection efficiency across samples, the Renilla luciferase expression vector pRLTK was included as an internal control. (B) Deletion mutants of the Rorg promoter were analyzed in the luciferase reporter assay with or without c-Rel co-transfection. Putative binding sites for c-Rel (Rel1 and Rel2) and NFAT are indicated. (C) WT and Rel or NFAT site-mutated Rorg promoter constructs were analyzed in the luciferase reporter assay with or without c-Rel co-transfection. The Rel1 site was mutated to TGGGACTCG (−201 to −193), the Rel2 site to CTGAAGTGC (−288 to −280), and the NFAT site to TTGTCAC (−96 to −90). The “X” indicates the mutated site. (D) Nuclear extracts were prepared from EL4 cells after stimulation for 6 h with PMA and ionomycin. Biotinylated Rorg Rel oligonucleotides or their mutants were absorbed by streptavidin-agarose beads, and then added to the nuclear extracts. The amount of c-Rel proteins in the precipitates were assessed by immunoblotting with anti–c-Rel. (E) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation conditions, as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel or control IgG. *, P
    Figure Legend Snippet: c-Rel binds to and activates the Rorg promoter through two specific Rel sites. (A) EL4 cells were transiently transfected with murine Rorg promoter luciferase construct together with an expression vector for full-length c-Rel, p65, p50, or RelB, or the empty vector as indicated. After 24 h, cells were treated with PMA and ionomycin for 5 h, and the luciferase activities measured. The promoter activity is presented as fold increase over cells transfected with empty vector. To normalize the transfection efficiency across samples, the Renilla luciferase expression vector pRLTK was included as an internal control. (B) Deletion mutants of the Rorg promoter were analyzed in the luciferase reporter assay with or without c-Rel co-transfection. Putative binding sites for c-Rel (Rel1 and Rel2) and NFAT are indicated. (C) WT and Rel or NFAT site-mutated Rorg promoter constructs were analyzed in the luciferase reporter assay with or without c-Rel co-transfection. The Rel1 site was mutated to TGGGACTCG (−201 to −193), the Rel2 site to CTGAAGTGC (−288 to −280), and the NFAT site to TTGTCAC (−96 to −90). The “X” indicates the mutated site. (D) Nuclear extracts were prepared from EL4 cells after stimulation for 6 h with PMA and ionomycin. Biotinylated Rorg Rel oligonucleotides or their mutants were absorbed by streptavidin-agarose beads, and then added to the nuclear extracts. The amount of c-Rel proteins in the precipitates were assessed by immunoblotting with anti–c-Rel. (E) Purified CD4 + T cells from 6-wk-old WT mice ( n = 3) were cultured under Th17 differentiation conditions, as described in Materials and methods. After 17 h, cells were fixed, and ChIP was performed using anti–c-Rel or control IgG. *, P

    Techniques Used: Transfection, Luciferase, Construct, Expressing, Plasmid Preparation, Activity Assay, Reporter Assay, Cotransfection, Binding Assay, Purification, Mouse Assay, Cell Culture, Chromatin Immunoprecipitation

    71) Product Images from "Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly"

    Article Title: Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx545

    Histone acetyltransferase 1 influences the recruitment of bromodomain proteins to nascent chromatin. ( A ) Detailed view of proteins depleted in absence of Hat1; arrows point to the chromatin modifiers containing bromodomains that were found depleted in absence of Hat1. ( B ) Peptides encoding the first 23 amino acids of histone H4 containing the modifications indicated across the top were incubated with recombinant Brg1 (top), Brd3 (middle) and Baz1a GST fusions. Each peptide was also separately incubated with a control GST protein. Protein–histone peptide complexes were then precipitated with streptavidin agarose beads. Proteins bound to the beads were then resolved by SDS-PAGE and visualized with anti-GST antibodies. INPUT lane contains the relative quantity of the GST control and protein GST fusion protein that were individually added to each reaction. ( C ) Histone peptide array (Epititan, Epicypher) was incubated with recombinant Brd3-GST fusion protein. Peptides bound by Brd3-GST were visualized and quantitated per manufactures instructions. The left panel is an image of the array probed with Brd3-GST. Peptides bound by Brd3 are labeld according to the legend (note that the subscript s or a refers to the symmetric or asymmetric methylation of the indicated arginine residue. The right panel table lists the modified peptides that were bound Brd3-GST.
    Figure Legend Snippet: Histone acetyltransferase 1 influences the recruitment of bromodomain proteins to nascent chromatin. ( A ) Detailed view of proteins depleted in absence of Hat1; arrows point to the chromatin modifiers containing bromodomains that were found depleted in absence of Hat1. ( B ) Peptides encoding the first 23 amino acids of histone H4 containing the modifications indicated across the top were incubated with recombinant Brg1 (top), Brd3 (middle) and Baz1a GST fusions. Each peptide was also separately incubated with a control GST protein. Protein–histone peptide complexes were then precipitated with streptavidin agarose beads. Proteins bound to the beads were then resolved by SDS-PAGE and visualized with anti-GST antibodies. INPUT lane contains the relative quantity of the GST control and protein GST fusion protein that were individually added to each reaction. ( C ) Histone peptide array (Epititan, Epicypher) was incubated with recombinant Brd3-GST fusion protein. Peptides bound by Brd3-GST were visualized and quantitated per manufactures instructions. The left panel is an image of the array probed with Brd3-GST. Peptides bound by Brd3 are labeld according to the legend (note that the subscript s or a refers to the symmetric or asymmetric methylation of the indicated arginine residue. The right panel table lists the modified peptides that were bound Brd3-GST.

    Techniques Used: Incubation, Recombinant, SDS Page, Peptide Microarray, Methylation, Modification

    72) Product Images from "Upregulation of CYP17A1 by Sp1-mediated DNA demethylation confers temozolomide resistance through DHEA-mediated protection in glioma"

    Article Title: Upregulation of CYP17A1 by Sp1-mediated DNA demethylation confers temozolomide resistance through DHEA-mediated protection in glioma

    Journal: Oncogenesis

    doi: 10.1038/oncsis.2017.31

    Increased Sp1-binding to the CYP17A1 promoter in TMZ-resistant glioma cells. ( a ) Cells were subjected to CHIP assays, and the indicated promoter fragment was amplified by PCR. ( b ) The CYP17A1 promoter sequence contains two putative Sp1-binding sites. ( c ) Sp1-binding activity was determined by DAPA. After incubation of protein lysates with the biotinylated Sp1-binding sequence (S1 or S2 probe) and streptavidin beads, the beads were analyzed by western blotting with an anti-Sp1 antibody. ( d ) Quantitative results for Sp1 expression and Sp1-binding activity. Data are expressed as the means±s.e.m. (* P
    Figure Legend Snippet: Increased Sp1-binding to the CYP17A1 promoter in TMZ-resistant glioma cells. ( a ) Cells were subjected to CHIP assays, and the indicated promoter fragment was amplified by PCR. ( b ) The CYP17A1 promoter sequence contains two putative Sp1-binding sites. ( c ) Sp1-binding activity was determined by DAPA. After incubation of protein lysates with the biotinylated Sp1-binding sequence (S1 or S2 probe) and streptavidin beads, the beads were analyzed by western blotting with an anti-Sp1 antibody. ( d ) Quantitative results for Sp1 expression and Sp1-binding activity. Data are expressed as the means±s.e.m. (* P

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction, Sequencing, Activity Assay, Incubation, Western Blot, Expressing

    73) Product Images from "Activation of Pre-mRNA Splicing by Human RNPS1 Is Regulated by CK2 Phosphorylation †"

    Article Title: Activation of Pre-mRNA Splicing by Human RNPS1 Is Regulated by CK2 Phosphorylation †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.25.4.1446-1457.2005

    Analyses of spliceosome complexes and RNA products by in vitro splicing assays with δ-crystallin pre-mRNA. (A) Schematic representation of the preparation of immobilized δ-crystallin pre-mRNA on streptavidin-agarose. B and AV indicate
    Figure Legend Snippet: Analyses of spliceosome complexes and RNA products by in vitro splicing assays with δ-crystallin pre-mRNA. (A) Schematic representation of the preparation of immobilized δ-crystallin pre-mRNA on streptavidin-agarose. B and AV indicate

    Techniques Used: In Vitro

    74) Product Images from "Identification of Plasmodium GAPDH epitopes for generation of antibodies that inhibit malaria infection"

    Article Title: Identification of Plasmodium GAPDH epitopes for generation of antibodies that inhibit malaria infection

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800111

    Three different mimotope peptides mimic different domains of the the P berghei GAPDH molecule. (A) The anti-P39 antibody specifically recognizes PbGAPDH in Western blots of P berghei sporozoite lysates. Ponceau stain and the anti-actin antibody were used as loading controls. “S”: lysates of sporozoites purified from infected A stephensi salivary glands; “M”: mock lysates obtained from uninfected A stephensi salivary glands. Anti-CSP antibody served as a positive control for the sporozoite lysate. A polyclonal anti-PbGAPDH antibody (red arrow) identifies a sporozoite GAPDH band with identical mobility to the anti-P39 band. (B) Antibodies against each of the mimotope peptides (P39, P61, and P52) recognize bands with identical mobility as PbGAPDH (red arrow), but not the pET tag protein (blue arrow). Each panel shows two lanes, the left containing the pET tag protein alone and the right the tagged recombinant PbGAPDH protein. The antibodies used to probe the blots are indicated at the bottom of each panel. The anti-tag and anti-KLH antibodies served as a positive and negative controls, respectively. The anti-mouse GAPDH antibody was used as a positive control for identification of the GAPDH protein. The position of the recombinant protein and tag proteins is indicated by arrows to the right. All data are representative of two independent experiments. (C) Each anti-peptide antibody specifically recognizes its own peptide. The biotinylated peptide indicated at the top of each panel was bound to the wells of streptavidin-coated ELISA plates and tested for binding by the antibodies denoted at the bottom of each panel. No evidence of cross-reaction was detected. All data are representative of two independent assays.
    Figure Legend Snippet: Three different mimotope peptides mimic different domains of the the P berghei GAPDH molecule. (A) The anti-P39 antibody specifically recognizes PbGAPDH in Western blots of P berghei sporozoite lysates. Ponceau stain and the anti-actin antibody were used as loading controls. “S”: lysates of sporozoites purified from infected A stephensi salivary glands; “M”: mock lysates obtained from uninfected A stephensi salivary glands. Anti-CSP antibody served as a positive control for the sporozoite lysate. A polyclonal anti-PbGAPDH antibody (red arrow) identifies a sporozoite GAPDH band with identical mobility to the anti-P39 band. (B) Antibodies against each of the mimotope peptides (P39, P61, and P52) recognize bands with identical mobility as PbGAPDH (red arrow), but not the pET tag protein (blue arrow). Each panel shows two lanes, the left containing the pET tag protein alone and the right the tagged recombinant PbGAPDH protein. The antibodies used to probe the blots are indicated at the bottom of each panel. The anti-tag and anti-KLH antibodies served as a positive and negative controls, respectively. The anti-mouse GAPDH antibody was used as a positive control for identification of the GAPDH protein. The position of the recombinant protein and tag proteins is indicated by arrows to the right. All data are representative of two independent experiments. (C) Each anti-peptide antibody specifically recognizes its own peptide. The biotinylated peptide indicated at the top of each panel was bound to the wells of streptavidin-coated ELISA plates and tested for binding by the antibodies denoted at the bottom of each panel. No evidence of cross-reaction was detected. All data are representative of two independent assays.

    Techniques Used: Western Blot, Staining, Purification, Infection, Positive Control, Positron Emission Tomography, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay

    75) Product Images from "Receptor-Like Protein Tyrosine Phosphatase ? Homodimerizes on the Cell Surface"

    Article Title: Receptor-Like Protein Tyrosine Phosphatase ? Homodimerizes on the Cell Surface

    Journal: Molecular and Cellular Biology

    doi:

    RPTPα appears to exist on the cell surface predominantly as homodimers. (A) 293 cells transiently expressing FL were surface biotinylated or not biotinylated. Cells were lysed, and biotinylated proteins were precipitated with streptavidin beads and separated by SDS-PAGE, and the biotinylated FL was detected by immunoblotting using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 . The blot was quantified using a PhosphorImager as described in Materials and Methods. Top and bottom panels are the immunoblot and quantification, respectively. The loading for each of the lanes was standardized using an equivalent amount of whole-cell lysate. Lane 7 is a longer exposure of lane 6. + or − biotin, labeled or not labeled with biotin; WCL, total whole-cell lysate; SN, whole-cell lysate supernatant after streptavidin bead precipitation; P, streptavidin precipitate. (B) BS 3 cross-linking was performed on 293 cells transiently transfected with either FL or FL.137C. Whole-cell lysates of cross-linked cells were separated by SDS-PAGE and probed using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 ).
    Figure Legend Snippet: RPTPα appears to exist on the cell surface predominantly as homodimers. (A) 293 cells transiently expressing FL were surface biotinylated or not biotinylated. Cells were lysed, and biotinylated proteins were precipitated with streptavidin beads and separated by SDS-PAGE, and the biotinylated FL was detected by immunoblotting using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 . The blot was quantified using a PhosphorImager as described in Materials and Methods. Top and bottom panels are the immunoblot and quantification, respectively. The loading for each of the lanes was standardized using an equivalent amount of whole-cell lysate. Lane 7 is a longer exposure of lane 6. + or − biotin, labeled or not labeled with biotin; WCL, total whole-cell lysate; SN, whole-cell lysate supernatant after streptavidin bead precipitation; P, streptavidin precipitate. (B) BS 3 cross-linking was performed on 293 cells transiently transfected with either FL or FL.137C. Whole-cell lysates of cross-linked cells were separated by SDS-PAGE and probed using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 ).

    Techniques Used: Expressing, SDS Page, Labeling, Transfection

    Mutations in the wedge diminish but do not abolish RPTPα oligomerization. (A) A schematic of RPTPα wedge mutant constructs, including point mutants FL.P210L.P211L and FL.E234A and deletion mutant Δ224-235. (B) For the top panel, transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα protein. For the bottom panel, transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. The bands representing FL.P210L.P211L dimers and Δ224-235 dimers are faint but detectable by PhosphorImager analysis. Biotinylation and cross-linking were done on parallel dishes from the same transfection. All the constructs were expressed to a similar level on the cell surface. (C) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTPα dimers over surface-expressed RPTPα, which were determined from the bottom and top portions of panel B, respectively, using a PhosphorImager. S, surface-expressed RPTPα (monomer).
    Figure Legend Snippet: Mutations in the wedge diminish but do not abolish RPTPα oligomerization. (A) A schematic of RPTPα wedge mutant constructs, including point mutants FL.P210L.P211L and FL.E234A and deletion mutant Δ224-235. (B) For the top panel, transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα protein. For the bottom panel, transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. The bands representing FL.P210L.P211L dimers and Δ224-235 dimers are faint but detectable by PhosphorImager analysis. Biotinylation and cross-linking were done on parallel dishes from the same transfection. All the constructs were expressed to a similar level on the cell surface. (C) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTPα dimers over surface-expressed RPTPα, which were determined from the bottom and top portions of panel B, respectively, using a PhosphorImager. S, surface-expressed RPTPα (monomer).

    Techniques Used: Mutagenesis, Construct, Transfection, Immunoprecipitation, SDS Page, Labeling

    Deletion of D2 diminishes but does not abolish RPTPα oligomerization. (A) A schematic of the D2 deletion mutant construct. (B) 293 cells transiently expressing ΔD2 protein were cross-linked or not cross-linked with BS 3 . Shown are the results of an immunoblotting analysis with anti-HA tag MAb 12CA5 on whole-cell lysates using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα protein. (D) Transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images obtained via PhosphorImager analysis. S/M, surface-expressed monomeric proteins.
    Figure Legend Snippet: Deletion of D2 diminishes but does not abolish RPTPα oligomerization. (A) A schematic of the D2 deletion mutant construct. (B) 293 cells transiently expressing ΔD2 protein were cross-linked or not cross-linked with BS 3 . Shown are the results of an immunoblotting analysis with anti-HA tag MAb 12CA5 on whole-cell lysates using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were immunoprecipitated with MAb 12CA5 to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα protein. (D) Transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images obtained via PhosphorImager analysis. S/M, surface-expressed monomeric proteins.

    Techniques Used: Mutagenesis, Construct, Expressing, Transfection, Immunoprecipitation, SDS Page, Labeling

    The ECD possesses relatively weak dimerization potential and is not required for the homodimerization of the full-length RPTPα. (A) A schematic of RPTPα constructs used in this figure. (B) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ECD.GPI (lanes 1 to 4) or ephrin A1 (lanes 5 to 7). Shown are the results of immunoblotting analysis with MAb 12CA5 of whole-cell lysates using ECL detection. (C) In the left panel, transiently transfected 293 cells were biotinylated. Streptavidin-agarose beads were used to isolate the total biotinylated surface proteins, which were then subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of surface-expressed RPTPα protein. In the right panel, transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. n.s., nonspecific band. (D) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ΔECD. Whole-cell lysates were subjected to immunoblotting analysis with anti-RPTPα serum 5478 using ECL detection.
    Figure Legend Snippet: The ECD possesses relatively weak dimerization potential and is not required for the homodimerization of the full-length RPTPα. (A) A schematic of RPTPα constructs used in this figure. (B) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ECD.GPI (lanes 1 to 4) or ephrin A1 (lanes 5 to 7). Shown are the results of immunoblotting analysis with MAb 12CA5 of whole-cell lysates using ECL detection. (C) In the left panel, transiently transfected 293 cells were biotinylated. Streptavidin-agarose beads were used to isolate the total biotinylated surface proteins, which were then subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of surface-expressed RPTPα protein. In the right panel, transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. n.s., nonspecific band. (D) Mock cross-linking and cross-linking on 293 cells transiently transfected with the construct ΔECD. Whole-cell lysates were subjected to immunoblotting analysis with anti-RPTPα serum 5478 using ECL detection.

    Techniques Used: Construct, Transfection, Labeling

    ΔCyto homodimerizes on the cell surface with high efficiency. (A) A schematic of the construct ΔCyto lacking the entire cytoplasmic domain. (B) 293 cells transiently transfected with FL or ΔCyto were treated or not treated with tunicamycin at 200 ng/ml and subsequently cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were precipitated with streptavidin beads to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα proteins. (D) Transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images from PhosphorImager analysis. S/M, surface-expressed monomeric proteins. (E) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTPα dimers over surface-expressed RPTPα, which were determined from panels C and D, respectively, using a PhosphorImager. n.s., nonspecific band.
    Figure Legend Snippet: ΔCyto homodimerizes on the cell surface with high efficiency. (A) A schematic of the construct ΔCyto lacking the entire cytoplasmic domain. (B) 293 cells transiently transfected with FL or ΔCyto were treated or not treated with tunicamycin at 200 ng/ml and subsequently cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis with MAb 12CA5 using ECL detection. (C) Transiently transfected 293 cells were biotinylated. Whole-cell lysates were precipitated with streptavidin beads to isolate the total RPTPα proteins, which were then subjected to SDS-PAGE and probed with 125 I-labeled streptavidin to determine the levels of surface-expressed RPTPα proteins. (D) Transiently transfected 293 cells were cross-linked with BS 3 . Whole-cell lysates were subjected to immunoblotting analysis using MAb 12CA5 followed by 125 I-labeled sheep anti-mouse IgG F(ab′) 2 to determine the levels of RPTPα dimers. Biotinylation (C) and cross-linking (D) were done on parallel dishes from the same transfection. Shown in panels C and D are images from PhosphorImager analysis. S/M, surface-expressed monomeric proteins. (E) Quantification of dimerization efficiency based on average of three replicates. The dimer/surface protein value is the ratio of the levels of RPTPα dimers over surface-expressed RPTPα, which were determined from panels C and D, respectively, using a PhosphorImager. n.s., nonspecific band.

    Techniques Used: Construct, Transfection, SDS Page, Labeling

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    Article Title: Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran
    Article Snippet: For preparation of biotin-streptavidin complexes, an excess of biotinylated protein was mixed with FITC-neutravidin on an end-over-end rotator at 4°C for several hours and then filtered for microinjection using 0.22-μm Millipore (Bedford, MA) centrifugal filtration units. .. Biotin-streptavidin complexes were microinjected as described below, and cells were processed for imaging by 3.7% formaldehyde fixation and mounting on glass slides.

    Motility Assay:

    Article Title: Two modes of microtubule sliding driven by cytoplasmic dynein
    Article Snippet: Paragraph title: In Vitro Motility Assay. ... For assays using anti-GFP antibody, assay chambers were sequentially coated with 1 mg/ml streptavidin, 1 mg/ml protein G-biotin (Sigma-Aldrich), 125 μg/ml anti-GFP antibody (3E6; Qbiogene, Irvine, CA), and ≈2 mg/ml α-casein ( , ).

    Sonication:

    Article Title: Polyphosphate is a cofactor for the activation of factor XI by thrombin
    Article Snippet: We made liposomes (20% phosphatidylserine, 40% phosphatidylcholine, 40% phosphatidylethanolamine [PCPSPE]; Avanti Polar Lipids) by sonication. .. Polybrene, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, phenylmethanesulfonyl fluoride (PMSF), streptavidin, theophylline, prostaglandin E1 (PGE1 ), thrombin receptor agonist peptide (SFLLRN-NH2 ), and hirudin were from Sigma-Aldrich.

    Recombinant:

    Article Title: Delivery of a Peptide Radiopharmaceutical to Brain with an IgG-Avidin Fusion Protein
    Article Snippet: .. The incubation was initiated by aspiration of medium, washing with Tris-buffered saline (TBS), and addition of 400 uL/well of TBS (pH=7.4) containing 0.5 uCi/mL of [3 H]-biotin, 0 or 1.0 ug/mL recombinant streptavidin (Sigma Chemical Co, St. Louis, MO), or 0 or 5.0 ug/mL cTfRMAb-AV fusion protein. .. The incubation at 37C was terminated at 2, 10, 30, 60, or 120 min by aspiration followed by washing of the monolayer 3 times with cold TBS.

    Article Title: Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly
    Article Snippet: Histone peptide binding assay Biotinylated histone peptides (Epicypher) were diluted to 1μM in 0.1% (v/v) triton-X100 in PBS and incubated with streptavidin beads (Novagen) for 3 h at room temperature. .. After three washes with binding buffer (50 mM Tris–HCl, pH 7.5, 250 mM NaCl, 0.05% (v/v) NP-40) peptide-bound beads were incubated with 100 nM GST-tagged recombinant proteins (Baz1a and Brg1, Epicypher; Brd3, Abcam) 3 h at room temperature.

    Article Title: Phosphoramidate End-labeling of Inorganic Polyphosphates: Facile Manipulation of Polyphosphate for Investigating and Modulating its Biological Activities †
    Article Snippet: .. Amine Surface and Carbo-BIND (hydrazide) multiwell strips were from Corning (Corning, NY); Nunc Immobilizer Streptavidin multiwell strips and Covalink-NH plates were from Thermo-Fisher (Waltham, MA); polystyrene coagulometer cuvettes were from Diagnostica Stago (Parsippany, NJ); amine-PEG2 -biotin was from Pierce (Rockford, IL, USA); polyethylenimine, spermidine, streptavidin, benzamidine and EDAC were from Sigma-Aldrich (St. Louis, MO); Cascade Blue-ethylenediamine was from Invitrogen (Carlsbad, CA); factor XIa, kallikrein, and thrombin were from Enzyme Research Laboratories (South Bend, IN); calf intestinal alkaline phosphatase was from Promega (Madison, WI); phospholipids were from Avanti Polar Lipids (Alabaster, AL); Biacore CM5 sensorchips were from GE Healthcare (Piscataway, NJ); chromogenic substrates S-2366 and S-2322 were from diaPharma (West Chester, OH); recombinant factor VIIa, and substrates Spectrozyme TH and Spectrozyme fVIIa were from American Diagnostica (Stamford, CT); and Sepabeads EC-HA were kindly provided by Resindion SRL (Milan, Italy). .. PolyP5, polyP25 and polyP45 (nominal mean polymer lengths, 5, 25 and 45, respectively, marketed as “sodium phosphate glass, types 5, 25 and 45”), and a heterodisperse preparation of very high MW polyP (marketed as “phosphate glass, water insoluble”) were from Sigma-Aldrich, as were sodium monophosphate, pyrophosphate, and triphosphate.

    Article Title: Molecular Analyses of DNA Helicases Involved in the Replicational Stress Response
    Article Snippet: .. Recombinant streptavidin was purchased as a lyophilized powder (Sigma) and dissolved in 25 mM HEPES (pH 7.4), 20% glycerol, and 10 mM NaCl. ..

    Article Title: Multi-Step Fibrinogen Binding to the Integrin αIIbβ3 Detected Using Force Spectroscopy
    Article Snippet: .. For experiments measuring streptavidin binding to biotin, recombinant streptavidin (Sigma, St. Louis, MO) was adsorbed onto pedestals whose surface was covalently coated with biotin-BSA to spatially orient the streptavidin molecules. .. Biotin-coated latex microspheres 1 μ m in diameter were purchased from Sigma.

    Article Title: Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran
    Article Snippet: BSA, recombinant myc-tagged NTF2 (in pET vector with N-terminal myc tag), and recombinant untagged NTF2 proteins (expressed and purified as described by ) were biotinylated using the Pierce (Rockford, IL) EZ-Link Sulfo-NHS-LC biotinylation kit according to the manufacturer's instructions. .. For preparation of biotin-streptavidin complexes, an excess of biotinylated protein was mixed with FITC-neutravidin on an end-over-end rotator at 4°C for several hours and then filtered for microinjection using 0.22-μm Millipore (Bedford, MA) centrifugal filtration units.

    Article Title: Two modes of microtubule sliding driven by cytoplasmic dynein
    Article Snippet: We carried out an in vitro motility assay by fixing the purified recombinant dynein constructs on a glass surface via streptavidin or anti-GFP antibody. .. For assays using anti-GFP antibody, assay chambers were sequentially coated with 1 mg/ml streptavidin, 1 mg/ml protein G-biotin (Sigma-Aldrich), 125 μg/ml anti-GFP antibody (3E6; Qbiogene, Irvine, CA), and ≈2 mg/ml α-casein ( , ).

    Article Title: A Sortase A programmable phage display format for improved panning of Fab antibody libraries
    Article Snippet: 25 or 50 ng of goat anti-human light chain antibodies, Streptavidin (Sigma-Aldrich), or Rat-anti- HA mAb 3F10 (Roche) were coated on a 96-well half-area ELISA plate (Costar) in 25 pL PBS. .. For coating of human ROR1, wells coated with streptavidin were washed once with 150 pL H2 0 and then 25 ng of recombinant biotinylated human ROR1 protein was added in 25 µL PBS.

    Article Title: IgE-FcεRI Interactions Determine HIV Coreceptor Usage and Susceptibility to Infection during Ontogeny of Mast Cells 1
    Article Snippet: Streptavidin (125 ng/ml; Sigma-Aldrich) was used in IgE-biotin cross-linking studies. .. Soluble SmEA was produced as described ( ) and HIV-1Ba-L gp120 recombinant protein (catalog no. 4961) was obtained from the National Institutes of Health AIDS Research & Reference Reagent Program (Germantown, MD).

    Article Title: Physiological protein blocks direct the Mre11–Rad50–Xrs2 and Sae2 nuclease complex to initiate DNA end resection
    Article Snippet: .. Where indicated, the substrate with reaction buffer was first incubated with 30 nM recombinant streptavidin (Sigma) per tetramer (in principle, 4 nM tetrameric streptavidin is sufficient to saturate 1 nM substrate with four biotin labels) or Ku (concentration as indicated) for 5 min at room temperature. ..

    Article Title: Polyphosphate is a cofactor for the activation of factor XI by thrombin
    Article Snippet: Polybrene, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, phenylmethanesulfonyl fluoride (PMSF), streptavidin, theophylline, prostaglandin E1 (PGE1 ), thrombin receptor agonist peptide (SFLLRN-NH2 ), and hirudin were from Sigma-Aldrich. .. EcPPXc, the recombinant polyP-binding domain of Escherichia coli exopolyphosphatase fused to maltose-binding protein and a His6 tag, was produced as described previously.

    Radioactivity:

    Article Title: Delivery of a Peptide Radiopharmaceutical to Brain with an IgG-Avidin Fusion Protein
    Article Snippet: The incubation was initiated by aspiration of medium, washing with Tris-buffered saline (TBS), and addition of 400 uL/well of TBS (pH=7.4) containing 0.5 uCi/mL of [3 H]-biotin, 0 or 1.0 ug/mL recombinant streptavidin (Sigma Chemical Co, St. Louis, MO), or 0 or 5.0 ug/mL cTfRMAb-AV fusion protein. .. The [3 H] radioactivity was determined with a Perkin-Elmer liquid scintillation counter using Ultima-Gold (Downers Grove, IL), and the monolayer protein was determined with the BCA assay.

    Fluorescence:

    Article Title: Multi-Step Fibrinogen Binding to the Integrin αIIbβ3 Detected Using Force Spectroscopy
    Article Snippet: The fluorescence of the glutaraldehyde-activated chamber did not change, whereas the adsorbed FITC-BSA was washed out of the control chamber. .. For experiments measuring streptavidin binding to biotin, recombinant streptavidin (Sigma, St. Louis, MO) was adsorbed onto pedestals whose surface was covalently coated with biotin-BSA to spatially orient the streptavidin molecules.

    Microscopy:

    Article Title: Two modes of microtubule sliding driven by cytoplasmic dynein
    Article Snippet: For assays using anti-GFP antibody, assay chambers were sequentially coated with 1 mg/ml streptavidin, 1 mg/ml protein G-biotin (Sigma-Aldrich), 125 μg/ml anti-GFP antibody (3E6; Qbiogene, Irvine, CA), and ≈2 mg/ml α-casein ( , ). .. MT sliding was observed under a BX-51 dark-field microscope (Olympus, Tokyo, Japan) with a ×40 objective lens.

    Purification:

    Article Title: Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran
    Article Snippet: BSA, recombinant myc-tagged NTF2 (in pET vector with N-terminal myc tag), and recombinant untagged NTF2 proteins (expressed and purified as described by ) were biotinylated using the Pierce (Rockford, IL) EZ-Link Sulfo-NHS-LC biotinylation kit according to the manufacturer's instructions. .. For preparation of biotin-streptavidin complexes, an excess of biotinylated protein was mixed with FITC-neutravidin on an end-over-end rotator at 4°C for several hours and then filtered for microinjection using 0.22-μm Millipore (Bedford, MA) centrifugal filtration units.

    Article Title: Two modes of microtubule sliding driven by cytoplasmic dynein
    Article Snippet: A purified dynein-containing solution (including 70 nM dynein construct, 1 mM ATP, and 0.1 mg/ml casein) was then perfused into the chambers twice, with a 5-min interval. .. For assays using anti-GFP antibody, assay chambers were sequentially coated with 1 mg/ml streptavidin, 1 mg/ml protein G-biotin (Sigma-Aldrich), 125 μg/ml anti-GFP antibody (3E6; Qbiogene, Irvine, CA), and ≈2 mg/ml α-casein ( , ).

    Article Title: Polyphosphate is a cofactor for the activation of factor XI by thrombin
    Article Snippet: Purified FXI, FXIa, FVa, β-thrombin, corn trypsin inhibitor (CTI), mouse anti-human FXI monoclonal antibody, and FXI- or FXII-deficient plasmas were from Haematologic Technologies. α-Thrombin was from Enzyme Research Laboratories. .. Polybrene, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, phenylmethanesulfonyl fluoride (PMSF), streptavidin, theophylline, prostaglandin E1 (PGE1 ), thrombin receptor agonist peptide (SFLLRN-NH2 ), and hirudin were from Sigma-Aldrich.

    Labeling:

    Article Title: Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran
    Article Snippet: Neutravidin, a carbohydrate-free derivative of streptavidin, was purchased from Pierce and labeled with FITC (Molecular Probes, Eugene, OR). .. For preparation of biotin-streptavidin complexes, an excess of biotinylated protein was mixed with FITC-neutravidin on an end-over-end rotator at 4°C for several hours and then filtered for microinjection using 0.22-μm Millipore (Bedford, MA) centrifugal filtration units.

    SDS Page:

    Article Title: Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran
    Article Snippet: Biotinylation was confirmed by SDS-PAGE and Western blotting using FITC-neutravidin as probe and visualization by fluorimaging. .. For preparation of biotin-streptavidin complexes, an excess of biotinylated protein was mixed with FITC-neutravidin on an end-over-end rotator at 4°C for several hours and then filtered for microinjection using 0.22-μm Millipore (Bedford, MA) centrifugal filtration units.

    Plasmid Preparation:

    Article Title: Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran
    Article Snippet: BSA, recombinant myc-tagged NTF2 (in pET vector with N-terminal myc tag), and recombinant untagged NTF2 proteins (expressed and purified as described by ) were biotinylated using the Pierce (Rockford, IL) EZ-Link Sulfo-NHS-LC biotinylation kit according to the manufacturer's instructions. .. For preparation of biotin-streptavidin complexes, an excess of biotinylated protein was mixed with FITC-neutravidin on an end-over-end rotator at 4°C for several hours and then filtered for microinjection using 0.22-μm Millipore (Bedford, MA) centrifugal filtration units.

    Enzyme-linked Immunosorbent Assay:

    Article Title: A Sortase A programmable phage display format for improved panning of Fab antibody libraries
    Article Snippet: .. 25 or 50 ng of goat anti-human light chain antibodies, Streptavidin (Sigma-Aldrich), or Rat-anti- HA mAb 3F10 (Roche) were coated on a 96-well half-area ELISA plate (Costar) in 25 pL PBS. .. For coating of human ROR1, wells coated with streptavidin were washed once with 150 pL H2 0 and then 25 ng of recombinant biotinylated human ROR1 protein was added in 25 µL PBS.

    Positron Emission Tomography:

    Article Title: Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran
    Article Snippet: BSA, recombinant myc-tagged NTF2 (in pET vector with N-terminal myc tag), and recombinant untagged NTF2 proteins (expressed and purified as described by ) were biotinylated using the Pierce (Rockford, IL) EZ-Link Sulfo-NHS-LC biotinylation kit according to the manufacturer's instructions. .. For preparation of biotin-streptavidin complexes, an excess of biotinylated protein was mixed with FITC-neutravidin on an end-over-end rotator at 4°C for several hours and then filtered for microinjection using 0.22-μm Millipore (Bedford, MA) centrifugal filtration units.

    In Vitro:

    Article Title: Two modes of microtubule sliding driven by cytoplasmic dynein
    Article Snippet: Paragraph title: In Vitro Motility Assay. ... For assays using anti-GFP antibody, assay chambers were sequentially coated with 1 mg/ml streptavidin, 1 mg/ml protein G-biotin (Sigma-Aldrich), 125 μg/ml anti-GFP antibody (3E6; Qbiogene, Irvine, CA), and ≈2 mg/ml α-casein ( , ).

    Produced:

    Article Title: IgE-FcεRI Interactions Determine HIV Coreceptor Usage and Susceptibility to Infection during Ontogeny of Mast Cells 1
    Article Snippet: Streptavidin (125 ng/ml; Sigma-Aldrich) was used in IgE-biotin cross-linking studies. .. Soluble SmEA was produced as described ( ) and HIV-1Ba-L gp120 recombinant protein (catalog no. 4961) was obtained from the National Institutes of Health AIDS Research & Reference Reagent Program (Germantown, MD).

    Article Title: Polyphosphate is a cofactor for the activation of factor XI by thrombin
    Article Snippet: Polybrene, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, phenylmethanesulfonyl fluoride (PMSF), streptavidin, theophylline, prostaglandin E1 (PGE1 ), thrombin receptor agonist peptide (SFLLRN-NH2 ), and hirudin were from Sigma-Aldrich. .. EcPPXc, the recombinant polyP-binding domain of Escherichia coli exopolyphosphatase fused to maltose-binding protein and a His6 tag, was produced as described previously.

    Concentration Assay:

    Article Title: Physiological protein blocks direct the Mre11–Rad50–Xrs2 and Sae2 nuclease complex to initiate DNA end resection
    Article Snippet: .. Where indicated, the substrate with reaction buffer was first incubated with 30 nM recombinant streptavidin (Sigma) per tetramer (in principle, 4 nM tetrameric streptavidin is sufficient to saturate 1 nM substrate with four biotin labels) or Ku (concentration as indicated) for 5 min at room temperature. ..

    Article Title: Polyphosphate is a cofactor for the activation of factor XI by thrombin
    Article Snippet: Note that all polyP concentrations are reported in this study in terms of phosphate monomer concentration (monomer formula, NaPO3 ), except for D that reports polyP polymer concentrations. .. Polybrene, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, phenylmethanesulfonyl fluoride (PMSF), streptavidin, theophylline, prostaglandin E1 (PGE1 ), thrombin receptor agonist peptide (SFLLRN-NH2 ), and hirudin were from Sigma-Aldrich.

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    Millipore streptavidin agarose beads
    Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with <t>streptavidin</t> beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).
    Streptavidin Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with streptavidin beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).

    Journal: Journal of Virology

    Article Title: Ubiquitin Ligase WWP1 Interacts with Ebola Virus VP40 To Regulate Egress

    doi: 10.1128/JVI.00812-17

    Figure Lengend Snippet: Proline-rich reading array screen and peptide pulldown. (A) Use of biotinylated eVP40 WT (MRRVILPTAPPEYMEAI[Lys-biotin]) peptide (50 μg) to screen a proline-rich reading array. The GST-WW domain fusion proteins are arrayed in duplicate and at different angles, as indicated in enlarged box C. Box C shows duplicate samples of all four WW domains from WWP1, WWP2, and ITCH as indicated. Additional positive interactions are indicated in the highlighted red boxes and ovals (A to H). The eVP40 mutant peptide (MRRVILPTAAAEAMEAI[Lys-biotin]) did not interact with any GST-WW domain fusion protein (data not shown). (B) Exogenously expressed FLAG-tagged WWP1-WT was pulled down with streptavidin beads bound to either eVP40 WT (WT) or PPXY mutant (mut) peptides and detected by Western blotting using anti-Flag antiserum (top). Expression controls for WWP1 and actin are shown (bottom).

    Article Snippet: Streptavidin agarose beads (Millipore) were prewashed once with 1× mild buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, 5 mM EGTA, 15 mM MgCl2 ), and 15 μg of the WT or PPXY mutant eVP40 peptide was incubated with the prewashed streptavidin beads in 500 μl of 1× mild buffer for 1 h at 4°C with rocking.

    Techniques: Mutagenesis, Western Blot, Expressing

    The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Journal: Glycobiology

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    doi: 10.1093/glycob/cwr097

    Figure Lengend Snippet: The rate of cell surface expression/appearance/transport of BRI2 is reduced in the absence of N-glycosylation. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. The newly synthesized proteins were labeled with 35 S in radiolabeling medium for 2 h (pulse) at 16°C and then were incubated in non-radiolabeling medium for 0′, 20′, 40′ and 60′ (chase). ( A ) Cell surface proteins were labeled with biotin and precipitated with streptavidin beads. Precipitated cell surface proteins were eluted from the beads and immunoprecipitated with 9B11 antibody against the myc epitope before electrophoresis and autoradiography. ( B ) Immunoprecipitation of cell extracts with 9B11, electrophoresis and autoradiography were performed to verify the expression levels of BRI2.

    Article Snippet: The cell extracts were centrifuged at 15,000 × g for 30 min and supernatants were incubated with 50 μL of streptavidin–agarose beads (Millipore) for 1 h at 4°C.

    Techniques: Expressing, Synthesized, Labeling, Radioactivity, Incubation, Immunoprecipitation, Electrophoresis, Autoradiography

    Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Journal: Glycobiology

    Article Title: Glycosylation of BRI2 on asparagine 170 is involved in its trafficking to the cell surface but not in its processing by furin or ADAM10

    doi: 10.1093/glycob/cwr097

    Figure Lengend Snippet: Inhibition of N-glycosylation of BRI2 inhibits its expression at the cell surface. Wild-type mycBRI2 or mycBRI2/N170A was expressed in HEK293 cells. Cell surface proteins were labeled with biotin (lanes 1 and 2) or were not labeled (lanes 3 and 4), as a control for biotinylation specificity. ( A ) Cell extracts were precipitated with streptavidin beads and analyzed with western blot against myc with 9B11 antibody. ( B ) Cell extracts were directly analyzed with western blot as a control for protein expression. The two immunoreactive bands of BRI2 proteins correspond to the furin-cleaved and the non-cleaved wild-type mycBRI2 or mycBRI2/N170A.

    Article Snippet: The cell extracts were centrifuged at 15,000 × g for 30 min and supernatants were incubated with 50 μL of streptavidin–agarose beads (Millipore) for 1 h at 4°C.

    Techniques: Inhibition, Expressing, Labeling, Western Blot

    A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins

    Journal: Journal of Virology

    Article Title: Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

    doi: 10.1128/JVI.01258-13

    Figure Lengend Snippet: A19 phosphorylation. (A) Western blot analysis. BS-C-1 cells were infected with vFS-A11 and vFS-A19 in the presence of 100 μCi/ml 32 P i . After 18 h, the cells were lysed, and the soluble extract was bound to streptavidin-agarose beads. Bound proteins

    Article Snippet: Soluble extracts obtained by low-speed centrifugation were allowed to bind to streptavidin-agarose beads (Millipore, Billerica, MA) for 3 h at 4°C.

    Techniques: Western Blot, Infection

    Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The

    Journal: Journal of Virology

    Article Title: Vaccinia Virus A19 Protein Participates in the Transformation of Spherical Immature Particles to Barrel-Shaped Infectious Virions

    doi: 10.1128/JVI.01258-13

    Figure Lengend Snippet: Expression and characterization of the A19 protein. (A) Schematic representation of the DNA construct used for generating recombinant vFS-A19. The FLAG- and streptavidin-binding peptide tag fused at the N terminus of the A19 ORF (FS) is indicated. The

    Article Snippet: Soluble extracts obtained by low-speed centrifugation were allowed to bind to streptavidin-agarose beads (Millipore, Billerica, MA) for 3 h at 4°C.

    Techniques: Expressing, Construct, Recombinant, Binding Assay

    Analysis of viral PPxY-host WW-domain interactions between BAG3 and LFV-Z by peptide pull-down assays. ( A ) Flow chart of the peptide pull-down assay using LFV-Z peptides and cell lysates expressing BAG3-WT; ( B ) Schematic diagram of BAG3-WT, BAG3-ΔN, and BAG3-ΔC mutants with the various domains highlighted in color and amino acid positions indicated; ( C ) Western blot of peptide pull-down assay using streptavidin agarose beads conjugated with either the LFV-Z WT or LFV-Z PY mutant peptide. BAG3 proteins were detected using anti-c-myc antibody (top blot). Expression controls for BAG3 and actin are shown in the bottom blot. These results are from 1 of 2 independent experiments.

    Journal: Diseases

    Article Title: Host Protein BAG3 is a Negative Regulator of Lassa VLP Egress

    doi: 10.3390/diseases6030064

    Figure Lengend Snippet: Analysis of viral PPxY-host WW-domain interactions between BAG3 and LFV-Z by peptide pull-down assays. ( A ) Flow chart of the peptide pull-down assay using LFV-Z peptides and cell lysates expressing BAG3-WT; ( B ) Schematic diagram of BAG3-WT, BAG3-ΔN, and BAG3-ΔC mutants with the various domains highlighted in color and amino acid positions indicated; ( C ) Western blot of peptide pull-down assay using streptavidin agarose beads conjugated with either the LFV-Z WT or LFV-Z PY mutant peptide. BAG3 proteins were detected using anti-c-myc antibody (top blot). Expression controls for BAG3 and actin are shown in the bottom blot. These results are from 1 of 2 independent experiments.

    Article Snippet: Briefly, extracts from HEK293T cells expressing either BAG3-WT, BAG3-ΔN, or BAG3-ΔC ( B) were incubated with streptavidin agarose beads bound with either the LFV-Z-WT or LFV-Z-mutant peptides.

    Techniques: Flow Cytometry, Pull Down Assay, Expressing, Western Blot, Mutagenesis