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    Structured Review

    Millipore streptavidin agarose beads
    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 <t>streptavidin-agarose</t> 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.
    Streptavidin Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    8) Product Images from "Coronavirus infectious bronchitis virus non-structural proteins 8 and 12 form stable complex independent of the non-translated regions of viral RNA and other viral proteins"

    Article Title: Coronavirus infectious bronchitis virus non-structural proteins 8 and 12 form stable complex independent of the non-translated regions of viral RNA and other viral proteins

    Journal: Virology

    doi: 10.1016/j.virol.2017.10.004

    Screen for IBV non-structural proteins that interact with the 5′-UTR (+), 5′-UTR (-) and 3′-UTR (+) of IBV. a. IBV nsp2, nsp5 and nsp10 showed binding activity to its 5′-UTR (+). Biotin pull-down assay of non-structural proteins with IBV 5′-UTR (+). Cell lysates of H1299 cells over-expressing different non-structural proteins were incubated with biotinylated 5′-UTR (+) probes and purified using streptavidin beads. Bound proteins were resolved by SDS-PAGE and western blot was performed. Proteins which did not express were not presented. C: cell lysate, E: elution. A representative result of two independent experiments was shown. b. IBV nsp5 and nsp10 showed binding activity to its 5′-UTR (-). Biotin pull-down assay of non-structural proteins with IBV 5′-UTR (-). Cell lysates of H1299 cells over-expressing different non-structural proteins were incubated with biotinylated 5′-UTR (-) probes and purified using streptavidin beads. Bound proteins were resolved by SDS-PAGE and western blot was performed. The expression of nsp2 was not detectable for this assay and was excluded. C: cell lysate, E: elution. A representative result of two independent experiments was shown. c. IBV nsp5, nsp8 and nsp9 showed binding activity to IBV 3′-UTR (+). Biotin pull-down assay of non-structural proteins with IBV 3′-UTR (+). Cell lysates of H1299 cells over-expressing different non-structural proteins were incubated with biotinylated 3′-UTR (+) probes and purified using streptavidin beads. Bound proteins were resolved by SDS-PAGE and western blot was performed. C: cell lysate, E: elution. A representative result of two independent experiments was shown.
    Figure Legend Snippet: Screen for IBV non-structural proteins that interact with the 5′-UTR (+), 5′-UTR (-) and 3′-UTR (+) of IBV. a. IBV nsp2, nsp5 and nsp10 showed binding activity to its 5′-UTR (+). Biotin pull-down assay of non-structural proteins with IBV 5′-UTR (+). Cell lysates of H1299 cells over-expressing different non-structural proteins were incubated with biotinylated 5′-UTR (+) probes and purified using streptavidin beads. Bound proteins were resolved by SDS-PAGE and western blot was performed. Proteins which did not express were not presented. C: cell lysate, E: elution. A representative result of two independent experiments was shown. b. IBV nsp5 and nsp10 showed binding activity to its 5′-UTR (-). Biotin pull-down assay of non-structural proteins with IBV 5′-UTR (-). Cell lysates of H1299 cells over-expressing different non-structural proteins were incubated with biotinylated 5′-UTR (-) probes and purified using streptavidin beads. Bound proteins were resolved by SDS-PAGE and western blot was performed. The expression of nsp2 was not detectable for this assay and was excluded. C: cell lysate, E: elution. A representative result of two independent experiments was shown. c. IBV nsp5, nsp8 and nsp9 showed binding activity to IBV 3′-UTR (+). Biotin pull-down assay of non-structural proteins with IBV 3′-UTR (+). Cell lysates of H1299 cells over-expressing different non-structural proteins were incubated with biotinylated 3′-UTR (+) probes and purified using streptavidin beads. Bound proteins were resolved by SDS-PAGE and western blot was performed. C: cell lysate, E: elution. A representative result of two independent experiments was shown.

    Techniques Used: Binding Assay, Activity Assay, Pull Down Assay, Expressing, Incubation, Purification, SDS Page, Western Blot

    9) 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

    10) 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

    11) Product Images from "Distinct functions of macrophage-derived and cancer cell-derived cathepsin Z combine to promote tumor malignancy via interactions with the extracellular matrix"

    Article Title: Distinct functions of macrophage-derived and cancer cell-derived cathepsin Z combine to promote tumor malignancy via interactions with the extracellular matrix

    Journal: Genes & Development

    doi: 10.1101/gad.249599.114

    Macrophage-derived CtsZ binds to cancer cells to enhance integrin-dependent invasion. ( A ) Cell surface expression and activity of CtsZ on wild-type (WT) and CtsZ −/− cancer cells. Wild-type and CtsZ −/− BTCs were exposed for 24 h to serum-free medium, wild-type macrophage CM, or CtsZ −/− macrophage CM prior to extensive washing and incubation with the cathepsin ABP DCG-04 (10 μmol/L) for 1 h at 4°C followed by additional PBS washes. Western blots of cell lysates using CtsZ- and CtsB-specific antibodies were developed with streptavidin peroxidase after performing pull-down purification of DCG-04-reactive polypeptides using streptavidin-agarose beads. Representative blots of three independent experiments are shown. ( B , C ) Wild-type and CtsZ −/− BTCs were grown and stimulated as described above prior to ice-cold ethanol fixation followed by cell surface staining of CtsZ. ( B ) Quantitation of CtsZ staining. ( C ) Representative images. Bars: all panels, 20 μm. ( D ) Invasion of wild-type BTCs stimulated with increasing concentrations of recombinant CtsZ was performed and monitored using xCELLigence. ( E ) Invasion of wild-type BTCs stimulated with either wild-type or CtsZ −/− macrophage CM in the presence of control IgG or blocking antibodies against integrins β1 or β3 was quantified using xCELLigence. (SFM) Serum-free medium. n = 4–6 replicate experiments. Graphs show mean ± SEM. P -values were obtained using unpaired two-tailed Student’s t -test; (*) P
    Figure Legend Snippet: Macrophage-derived CtsZ binds to cancer cells to enhance integrin-dependent invasion. ( A ) Cell surface expression and activity of CtsZ on wild-type (WT) and CtsZ −/− cancer cells. Wild-type and CtsZ −/− BTCs were exposed for 24 h to serum-free medium, wild-type macrophage CM, or CtsZ −/− macrophage CM prior to extensive washing and incubation with the cathepsin ABP DCG-04 (10 μmol/L) for 1 h at 4°C followed by additional PBS washes. Western blots of cell lysates using CtsZ- and CtsB-specific antibodies were developed with streptavidin peroxidase after performing pull-down purification of DCG-04-reactive polypeptides using streptavidin-agarose beads. Representative blots of three independent experiments are shown. ( B , C ) Wild-type and CtsZ −/− BTCs were grown and stimulated as described above prior to ice-cold ethanol fixation followed by cell surface staining of CtsZ. ( B ) Quantitation of CtsZ staining. ( C ) Representative images. Bars: all panels, 20 μm. ( D ) Invasion of wild-type BTCs stimulated with increasing concentrations of recombinant CtsZ was performed and monitored using xCELLigence. ( E ) Invasion of wild-type BTCs stimulated with either wild-type or CtsZ −/− macrophage CM in the presence of control IgG or blocking antibodies against integrins β1 or β3 was quantified using xCELLigence. (SFM) Serum-free medium. n = 4–6 replicate experiments. Graphs show mean ± SEM. P -values were obtained using unpaired two-tailed Student’s t -test; (*) P

    Techniques Used: Derivative Assay, Expressing, Activity Assay, Incubation, Western Blot, Purification, Staining, Quantitation Assay, Recombinant, Blocking Assay, Two Tailed Test

    12) 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

    13) 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

    14) 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

    15) 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

    16) Product Images from "COP9 Signalosome Component JAB1/CSN5 Is Necessary for T Cell Signaling through LFA-1 and HIV-1 Replication"

    Article Title: COP9 Signalosome Component JAB1/CSN5 Is Necessary for T Cell Signaling through LFA-1 and HIV-1 Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041725

    Pep24 specifically binds to JAB1. The indicated biotin-conjugated peptides were incubated with GST or with the GST-fused JAB1. The complex of peptide-GST fusion protein immobilized on streptavidin-agarose was detected by western blotting with anti-GST antibody.
    Figure Legend Snippet: Pep24 specifically binds to JAB1. The indicated biotin-conjugated peptides were incubated with GST or with the GST-fused JAB1. The complex of peptide-GST fusion protein immobilized on streptavidin-agarose was detected by western blotting with anti-GST antibody.

    Techniques Used: Incubation, Western Blot

    17) Product Images from "Elucidating combinatorial histone modifications and crosstalks by coupling histone-modifying enzyme with biotin ligase activity"

    Article Title: Elucidating combinatorial histone modifications and crosstalks by coupling histone-modifying enzyme with biotin ligase activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1247

    Affinity purification of BirA-biotinylated and MSK1-modified nucleosomes. ( A ) Workflow of the affinity purification method used for isolation of co-modified nucleosomes. ( B ) DNA was extracted from MNase-digested input chromatin, resolved on a 2% agarose gel and visualized with ethidium bromide. ( C ) Mono-nucleosomes containing H3.3-AviFlag were purified as described in (A). Input chromatin (left) and pull-down material (right) were resolved on SDS-PAGE gels and visualized by coomassie blue staining (top) or analysed by Western blotting using the indicated antibodies (bottom). * streptavidin monomer.
    Figure Legend Snippet: Affinity purification of BirA-biotinylated and MSK1-modified nucleosomes. ( A ) Workflow of the affinity purification method used for isolation of co-modified nucleosomes. ( B ) DNA was extracted from MNase-digested input chromatin, resolved on a 2% agarose gel and visualized with ethidium bromide. ( C ) Mono-nucleosomes containing H3.3-AviFlag were purified as described in (A). Input chromatin (left) and pull-down material (right) were resolved on SDS-PAGE gels and visualized by coomassie blue staining (top) or analysed by Western blotting using the indicated antibodies (bottom). * streptavidin monomer.

    Techniques Used: Affinity Purification, Modification, Isolation, Agarose Gel Electrophoresis, Purification, SDS Page, Staining, Western Blot

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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

    25) 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

    26) 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

    27) 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

    28) 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

    29) 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

    30) 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

    31) 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

    32) 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

    33) Product Images from "Maturation of BRI2 generates a specific inhibitor that reduces APP processing at the plasma membrane and in endocytic vesicles"

    Article Title: Maturation of BRI2 generates a specific inhibitor that reduces APP processing at the plasma membrane and in endocytic vesicles

    Journal: Neurobiology of aging

    doi: 10.1016/j.neurobiolaging.2009.08.005

    BRI2 inhibits APP processing on the plasma membrane and in endocytic vesicles. (A) HeLa cells transfected with APP or APP plus Flag-BRI2 were biotinylated, incubated at 37 °C for the indicated times, lysed and precipitated with streptavidin beads.
    Figure Legend Snippet: BRI2 inhibits APP processing on the plasma membrane and in endocytic vesicles. (A) HeLa cells transfected with APP or APP plus Flag-BRI2 were biotinylated, incubated at 37 °C for the indicated times, lysed and precipitated with streptavidin beads.

    Techniques Used: Transfection, Incubation

    34) 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

    35) 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

    36) 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

    37) 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

    38) 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

    39) 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

    40) 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

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    Isolation:

    Article Title: The involvement of replication in single stranded oligonucleotide-mediated gene repair
    Article Snippet: .. After isolation of the plasmid DNA, biotinylated DNA was captured on streptavidin-conjugated magnetic agarose beads (Novagen), and was purified as described by Edwards et al . ( ). .. The identity of the biotinylated pmKan plasmid was confirmed by PCR amplification of a 496 bp-region of the kan gene which contained the site of the original point mutation using primer 1 and primer 2.

    Article Title: Insulin-like growth factor-I receptor is suppressed through transcriptional repression and mRNA destabilization by a novel energy restriction-mimetic agent
    Article Snippet: .. Protein-biotinylated RNA complexes were isolated with streptavidin-Sepharose (Sigma–Aldrich) at 4°C for 2h with rotation. .. After washing with binding buffer (20mM Tris–HCl pH 7.5, 150mM NaCl and 1% Triton X-100), the complexes were resuspended in 2× SDS sample buffer (100mM Tris–HCl pH 6.8, 4% SDS, 5% beta-mercaptoethanol, 20% glycerol and 0.1% bromophenol blue), boiled for 10min, resolved by 10% SDS–polyacrylamide gel and subjected to immunoblotting with anti-HuR antibody.

    Purification:

    Article Title: The involvement of replication in single stranded oligonucleotide-mediated gene repair
    Article Snippet: .. After isolation of the plasmid DNA, biotinylated DNA was captured on streptavidin-conjugated magnetic agarose beads (Novagen), and was purified as described by Edwards et al . ( ). .. The identity of the biotinylated pmKan plasmid was confirmed by PCR amplification of a 496 bp-region of the kan gene which contained the site of the original point mutation using primer 1 and primer 2.

    Immunoprecipitation:

    Article Title: In situ imaging and isolation of proteins using dsDNA oligonucleotides
    Article Snippet: .. The precleared lysate was then incubated overnight at 4°C with continuous mixing with either 100 μl of streptavidin sepharose alone (Mock) or pre-incubated with O-Sym oligo or 50 μl of Protein G sepharose pre-incubated with 20 μg of anti-Flag epitope antibody M2 (Sigma) (immunoprecipitation or IP). .. In addition, unrelated dsDNA oligo (i.e. TetO) was added to a concentration of ∼1 μM to the precleared lysate during protein isolation to reduce the non-specific co-purification of other DNA binding proteins.

    Article Title: Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein
    Article Snippet: .. After lysis of the cells with radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore), biotinylated proteins were immunoprecipitated using Streptavidin Mag Sepharose (Sigma) and then SDS-PAGE under nonreducing conditions. .. The presence of RSV F proteins was detected by transferring the gel to a polyvinylidene difluoride (PVDF) membrane and subsequent incubation with RSV F-specific MAb.

    Incubation:

    Article Title: In situ imaging and isolation of proteins using dsDNA oligonucleotides
    Article Snippet: .. The precleared lysate was then incubated overnight at 4°C with continuous mixing with either 100 μl of streptavidin sepharose alone (Mock) or pre-incubated with O-Sym oligo or 50 μl of Protein G sepharose pre-incubated with 20 μg of anti-Flag epitope antibody M2 (Sigma) (immunoprecipitation or IP). .. In addition, unrelated dsDNA oligo (i.e. TetO) was added to a concentration of ∼1 μM to the precleared lysate during protein isolation to reduce the non-specific co-purification of other DNA binding proteins.

    Article Title: Ribonucleoprotein particles of bacterial small non-coding RNA IsrA (IS61 or McaS) and its interaction with RNA polymerase core may link transcription to mRNA fate
    Article Snippet: .. RNAP was eluted from streptavidin sepharose by incubation with HRV3C (Sigma) for 16 h at 4°C in BB @150mM NaCl (in the presence of Superasin) and removed from nickel sepharose by digestion with proteinase K (Fluka). .. RNA was isolated from eluates by phenol–chloroform (Sigma), and chloroform (Sigma) extractions followed by ethanol precipitation in the presence of glycogen (Roche) as a carrier.

    Lysis:

    Article Title: Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein
    Article Snippet: .. After lysis of the cells with radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore), biotinylated proteins were immunoprecipitated using Streptavidin Mag Sepharose (Sigma) and then SDS-PAGE under nonreducing conditions. .. The presence of RSV F proteins was detected by transferring the gel to a polyvinylidene difluoride (PVDF) membrane and subsequent incubation with RSV F-specific MAb.

    SDS Page:

    Article Title: Antibody-Induced Internalization of the Human Respiratory Syncytial Virus Fusion Protein
    Article Snippet: .. After lysis of the cells with radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore), biotinylated proteins were immunoprecipitated using Streptavidin Mag Sepharose (Sigma) and then SDS-PAGE under nonreducing conditions. .. The presence of RSV F proteins was detected by transferring the gel to a polyvinylidene difluoride (PVDF) membrane and subsequent incubation with RSV F-specific MAb.

    Plasmid Preparation:

    Article Title: The involvement of replication in single stranded oligonucleotide-mediated gene repair
    Article Snippet: .. After isolation of the plasmid DNA, biotinylated DNA was captured on streptavidin-conjugated magnetic agarose beads (Novagen), and was purified as described by Edwards et al . ( ). .. The identity of the biotinylated pmKan plasmid was confirmed by PCR amplification of a 496 bp-region of the kan gene which contained the site of the original point mutation using primer 1 and primer 2.

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    Millipore streptavidin agarose beads
    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 <t>streptavidin</t> 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.
    Streptavidin Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    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