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  • 99
    Millipore sds polyacrylamide gels
    Lobeline treatment increases laminin binding. Lysates of surface biotinylated wild type ( wt ) ( A ) or <t>mdx</t> myotubes ( B ) treated with lobeline ( L ) or control ( C ) were separated by <t>SDS-PAGE</t> and blotted, laminin was incubated with the blots, and bound laminin was detected with anti-laminin. Blots were stripped and reprobed with mAb IIH6.
    Sds Polyacrylamide Gels, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12966 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Bio-Rad polyacrylamide gel
    Lobeline treatment increases laminin binding. Lysates of surface biotinylated wild type ( wt ) ( A ) or <t>mdx</t> myotubes ( B ) treated with lobeline ( L ) or control ( C ) were separated by <t>SDS-PAGE</t> and blotted, laminin was incubated with the blots, and bound laminin was detected with anti-laminin. Blots were stripped and reprobed with mAb IIH6.
    Polyacrylamide Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 13305 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sds polyacrylamide gel
    Arginines (R44, R53 and R57) in the recognition helix and N-terminal arm (R3 and R5) of the homeodomain of Pax6 are important for the interaction with the paired domain. <t>GST</t> pull-down assays with Pax6 HD, and HD mutations fused to GST and immobilized on glutathione–agarose beads and in vitro translated Pax6ΔHD. ( A ) The GST pull-downs were performed as described in the legend to Figure 1 . The panel shows two different experiments using two different GST-HD fusions. The GST-HD protein (upper panel) contains two amino acids N-terminal to the HD while the GST-18L-HD (lower panel) contains 18 amino acids of the linker region N-terminal to the HD to study the effect of mutating also at −3 relative to the start of the HD. ( B ) Quantitative representation of the interaction data. A Fuji Bio-imaging analyzer (BAS5000) equipped with Image Gauge version 4.0 software was used to quantitate 35 S-labeled proteins in the <t>SDS–polyacrylamide</t> gels. The amount 35 S-labeled Pax6ΔHD pulled down by wild-type GST-HD was set to 100%. The data shown represent the mean of three independent experiments.
    Sds Polyacrylamide Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 8441 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore polyacrylamide gel
    Arginines (R44, R53 and R57) in the recognition helix and N-terminal arm (R3 and R5) of the homeodomain of Pax6 are important for the interaction with the paired domain. <t>GST</t> pull-down assays with Pax6 HD, and HD mutations fused to GST and immobilized on glutathione–agarose beads and in vitro translated Pax6ΔHD. ( A ) The GST pull-downs were performed as described in the legend to Figure 1 . The panel shows two different experiments using two different GST-HD fusions. The GST-HD protein (upper panel) contains two amino acids N-terminal to the HD while the GST-18L-HD (lower panel) contains 18 amino acids of the linker region N-terminal to the HD to study the effect of mutating also at −3 relative to the start of the HD. ( B ) Quantitative representation of the interaction data. A Fuji Bio-imaging analyzer (BAS5000) equipped with Image Gauge version 4.0 software was used to quantitate 35 S-labeled proteins in the <t>SDS–polyacrylamide</t> gels. The amount 35 S-labeled Pax6ΔHD pulled down by wild-type GST-HD was set to 100%. The data shown represent the mean of three independent experiments.
    Polyacrylamide Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher polyacrylamide gel
    Arginines (R44, R53 and R57) in the recognition helix and N-terminal arm (R3 and R5) of the homeodomain of Pax6 are important for the interaction with the paired domain. <t>GST</t> pull-down assays with Pax6 HD, and HD mutations fused to GST and immobilized on glutathione–agarose beads and in vitro translated Pax6ΔHD. ( A ) The GST pull-downs were performed as described in the legend to Figure 1 . The panel shows two different experiments using two different GST-HD fusions. The GST-HD protein (upper panel) contains two amino acids N-terminal to the HD while the GST-18L-HD (lower panel) contains 18 amino acids of the linker region N-terminal to the HD to study the effect of mutating also at −3 relative to the start of the HD. ( B ) Quantitative representation of the interaction data. A Fuji Bio-imaging analyzer (BAS5000) equipped with Image Gauge version 4.0 software was used to quantitate 35 S-labeled proteins in the <t>SDS–polyacrylamide</t> gels. The amount 35 S-labeled Pax6ΔHD pulled down by wild-type GST-HD was set to 100%. The data shown represent the mean of three independent experiments.
    Polyacrylamide Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 6595 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore sodium dodecyl sulfate polyacrylamide gel
    Production of Nb displaying LVs. ( a ) Non-modified HEK 293T cells or HEK 293T cells stably expressing Nb BCII10 or DC2.1 were used to produce LVs pseudotyped with VSV.G or VSV.GS, respectively. Three days after transfection of these cells with the VSV.G or VSV.GS, gag/pol and transgene encoding plasmids, we evaluated the expression of the transgene (Thy1.1 or tNGFR), as well as the Nb (myc tag) by flow cytometry. Non-transfected HEK 293T cells served as a control. The flow cytometry dot plots demonstrate high expression of the transgene ( y axis) in all transfected cells (VSV.G, BCII10 and DC2.1) and high expression of Nbs ( x -axis) on the Nb-modified HEK 293T cells (BCII10 and DC2.1). One representative experiment is shown ( n =6). ( b ) To compare the LV preparations we determined their RT content. The graph depicts the amount of RT (ng RT/μl) in the LV preparations. Each dot represents one LV stock, the horizontal line shows the mean ( n =6). ( c ) An ELISA involving anti-VHH and anti-Thy1.1 as capture and detection antibodies, respectively, was used to demonstrate the incorporation of Nbs into the surface of Thy1.1 encoding LVs. A serial dilution of LVs was applied (5, 2.5 and 1.25 ng RT). The graph depicts the OD-values detected. One representative experiment is shown ( n =3). ( d ) Western blot was performed as a quality control of the LVs. After separation on a 15% <t>sodium</t> <t>dodecyl</t> <t>sulphate-polyacrylamide</t> <t>gel</t> and transfer to a nitrocellulose membrane, the Nbs (±25 kDa) and VSV.GS (±15 kDa), which both contain an HA epitope tag, were detected with an anti-HA antibody. One representative experiment is shown ( n =4). ( e ) The density of the western blot signals was determined using the Photocapt MW software and used to determine the ratio of Nbs/VSV.GS on the LVs. This ratio is shown in the graph, in which each dot represents one LV stock and the horizontal line shows the mean ( n =4).
    Sodium Dodecyl Sulfate Polyacrylamide Gel, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1877 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad sodium dodecyl sulfate polyacrylamide gel
    Identification of NFM as the target antigen of non-NMDAR autoantibodies. We cotransfected HEK293T cells with GFP and NFM, NFH, NFL, or βIII-tubulin (A, column 1); column 2 shows the immunostaining for a commercially available antibody in red, and column 3 shows the merged reactivities. The non-NMDAR autoantibodies in the serum from patients reacted with HEK293T cells expressing human NFM in a fluorescence-based cell-binding assay (B). From top to bottom: binding of antibodies from patients' serum or CSF using an antihuman immunoglobulin G secondary antibody (red) to cells transfected with human NFM (green), merged images (yellow), and the absence of staining of nontransfected cells. Patients with anti-NMDAR encephalitis were negative for NFH, NFL, and βIII-tubulin antibodies, while cells displayed filamentous patterns. No binding was observed when using healthy serum. Representative results obtained by Western blotting (“P” and “C” indicate specific patients and controls, respectively) (C). Whole-cell lysates prepared from human HEK293T cells with (+) or without (−) transfection of the human NFM gene were subjected to <t>sodium</t> <t>dodecyl</t> <t>sulfate–polyacrylamide</t> <t>gel</t> electrophoresis. The blots were exposed to serum from seven patients with anti-NMDAR encephalitis and seven healthy controls (C1–C3) after a total of 20 µg of protein lysates from HEK293T cells. Antibodies that bound to the HEK293T cell lysates transfected with NFM that predominantly reacted with one antigen of approximately 160 kDa were seen in seru m (diluted 1:100) from patients 3, 13, 24, 39, 43, 46, and 51. This binding was not observed in controls. No protein bands against the HEK293T cells without transfection (as negative controls) were detected in any of the samples. NFM was detected using commercially available anti-NFM antibodies as a positive control. Scale bar=20 µm. CSF: cerebrospinal fluid, GFP: green fluorescent protein, HEK: human embryonic kidney, NFH: heavy neurofilament, NFL: light neurofilament, NFM: medium neurofilament, NMDAR: N-methyl-D-aspartate receptor.
    Sodium Dodecyl Sulfate Polyacrylamide Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 1500 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher bis tris polyacrylamide gel
    <t>CPV</t> purification and characterization. A. Sucrose gradient purification . VLPs preparation from infected cell culture lysates purified by sucrose gradient centrifugation (10–40%). Bands of CPV-VLPs that were derivatized with OG-488 are visible in the gradient just above the middle of the tube (left panel) and appear fluorescent green under a UV-light source (right panel). B. SDS-PAGE analyses . The purified VLPs were subjected to electrophoresis in 4–12% <t>Bis-tris</t> gel and stained with SimplyBlue (Invitrogen) to reveal the proteins (left panel). The Seeblue plus protein molecular weight standards in kDa (Invitrogen) are indicated on the side of the gel picture (lane 1). Lanes 2 and 3 contain protein from CPV-VLPs derivatized with OG-488 and CPV-VLPs respectively. Prior to staining, the gel (right panel) visualized with a UV-light source showed a fluorescent 62 kDa band in the lane of OG-488 derivatized CPV-VLPs (lane 2f) and lacked any fluorescent bands in the native CPV-VLPs (lane 3f).
    Bis Tris Polyacrylamide Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1640 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher sds polyacrylamide gel
    Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% <t>SDS-PAGE</t> gels and transferred to <t>PVDF</t> membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments
    Sds Polyacrylamide Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad polyacrylamide gradient gels
    Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% <t>SDS-PAGE</t> gels and transferred to <t>PVDF</t> membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments
    Polyacrylamide Gradient Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 938 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Bio-Rad precast polyacrylamide gel
    Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% <t>SDS-PAGE</t> gels and transferred to <t>PVDF</t> membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments
    Precast Polyacrylamide Gel, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 694 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM polyacrylamide gel
    Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% <t>SDS-PAGE</t> gels and transferred to <t>PVDF</t> membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments
    Polyacrylamide Gel, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 1041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher gradient polyacrylamide gel
    Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% <t>SDS-PAGE</t> gels and transferred to <t>PVDF</t> membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments
    Gradient Polyacrylamide Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 866 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schuell GmbH polyacrylamide gel
    Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% <t>SDS-PAGE</t> gels and transferred to <t>PVDF</t> membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments
    Polyacrylamide Gel, supplied by Schuell GmbH, used in various techniques. Bioz Stars score: 92/100, based on 596 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology sds polyacrylamide gel
    POLA1 levels are elevated in colorectal cancer and ST1926 decreases its expression. A. ST1926 reduces POLA1 protein levels in colorectal cell lines. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to two days. Total <t>SDS</t> protein lysates (80 µg/lane) were <t>immunoblotted</t> against POLA1 antibody. Similar trends in protein levels were observed in two independent experiments. B. POLA1 basal protein levels are elevated in CRC cell lines compared to the normal-like NCM460 cells. Total SDS protein lysates (80 µg/lane) were immunoblotted against POLA1 antibody. Similar trends were observed in two independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. Expression levels of POLA1 ]. D. POLA1 ]. P values
    Sds Polyacrylamide Gel, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 574 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Schuell GmbH sds polyacrylamide gel
    Immunoblotting with <t>anti-Lyt51</t> antibodies. Lane 1, purified six-His-tagged Lyt51; lane 2, DN-001065; lane 3, mitomycin C-induced DN-001065; lane 4, CNRZ 302. The <t>SDS</t> cell extracts were prepared 40 min after mitomycin C induction of strain DN-001065. For each cell extract, the same protein amount was transferred on a nitrocellulose membrane. The blot was incubated with the purified antibodies directed against the six-His-tagged Lyt51 and then with protein G coupled to horseradish peroxidase. Positions of the six-His-tagged Lyt51 and the 31-kDa protein reacting with the anti-Lyt51 antibodies are indicated by arrowheads.
    Sds Polyacrylamide Gel, supplied by Schuell GmbH, used in various techniques. Bioz Stars score: 92/100, based on 711 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Lobeline treatment increases laminin binding. Lysates of surface biotinylated wild type ( wt ) ( A ) or mdx myotubes ( B ) treated with lobeline ( L ) or control ( C ) were separated by SDS-PAGE and blotted, laminin was incubated with the blots, and bound laminin was detected with anti-laminin. Blots were stripped and reprobed with mAb IIH6.

    Journal: The Journal of Biological Chemistry

    Article Title: High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding *

    doi: 10.1074/jbc.M111.334581

    Figure Lengend Snippet: Lobeline treatment increases laminin binding. Lysates of surface biotinylated wild type ( wt ) ( A ) or mdx myotubes ( B ) treated with lobeline ( L ) or control ( C ) were separated by SDS-PAGE and blotted, laminin was incubated with the blots, and bound laminin was detected with anti-laminin. Blots were stripped and reprobed with mAb IIH6.

    Article Snippet: Wild type and mdx muscle cell eluates were separated on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore).

    Techniques: Binding Assay, SDS Page, Incubation

    Lobeline increases abundance of multiple muscle cell proteins. A , wild type ( wt ) primary myoblasts differentiated in the presence of 100 μ m lobeline ( L ) or control ( C ) were solubilized in radioimmune precipitation assay buffer, and 60 μg of protein was separated by SDS-PAGE and blotted to nitrocellulose. Membranes were probed with antibodies against agrin, dystrophin, utrophin, dystroglycans (α- and β-DG), sarcospan ( SSPN ), sarcoglycans (α-, β-, and γ-SG), β1D integrin, dysferlin, and caveolin-3. GAPDH was a loading control. Lobeline treatment increased agrin, integrin, dysferlin, DGC, and UGC protein levels. B , mdx primary myoblasts were treated as described in A . Membranes were probed similarly with the addition of antibody to β-SG. Dystrophin protein was not detected due to mutation in the dystrophin gene in mdx cells. Increases were observed for agrin, dysferlin, utrophin, and α- and β-dystroglycans.

    Journal: The Journal of Biological Chemistry

    Article Title: High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding *

    doi: 10.1074/jbc.M111.334581

    Figure Lengend Snippet: Lobeline increases abundance of multiple muscle cell proteins. A , wild type ( wt ) primary myoblasts differentiated in the presence of 100 μ m lobeline ( L ) or control ( C ) were solubilized in radioimmune precipitation assay buffer, and 60 μg of protein was separated by SDS-PAGE and blotted to nitrocellulose. Membranes were probed with antibodies against agrin, dystrophin, utrophin, dystroglycans (α- and β-DG), sarcospan ( SSPN ), sarcoglycans (α-, β-, and γ-SG), β1D integrin, dysferlin, and caveolin-3. GAPDH was a loading control. Lobeline treatment increased agrin, integrin, dysferlin, DGC, and UGC protein levels. B , mdx primary myoblasts were treated as described in A . Membranes were probed similarly with the addition of antibody to β-SG. Dystrophin protein was not detected due to mutation in the dystrophin gene in mdx cells. Increases were observed for agrin, dysferlin, utrophin, and α- and β-dystroglycans.

    Article Snippet: Wild type and mdx muscle cell eluates were separated on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore).

    Techniques: SDS Page, Mutagenesis

    Identification of WFA binding glycoproteins in control-treated versus lobeline-treated cells. A , equal amounts of cell protein from lysates of C2C12 myotubes treated with 100 μ m lobeline ( L ) or control ( C ) for 48 h were precipitated with WFA beads, bound proteins were eluted with GalNAc, eluted proteins were separated by SDS-PAGE, and blots were probed with WFA or mAb IIH6. Equal amounts of cell protein from lysates of wild type ( wt ) ( B ) or mdx myotubes ( C ) treated with lobeline or control were precipitated with WFA beads, bound proteins were eluted with GalNAc, eluted proteins were separated by SDS-PAGE, and blots were probed with WFA or mAbs IIH6 or MANDAG2 ( MA ), which recognizes β-DG. Results are representative of four independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding *

    doi: 10.1074/jbc.M111.334581

    Figure Lengend Snippet: Identification of WFA binding glycoproteins in control-treated versus lobeline-treated cells. A , equal amounts of cell protein from lysates of C2C12 myotubes treated with 100 μ m lobeline ( L ) or control ( C ) for 48 h were precipitated with WFA beads, bound proteins were eluted with GalNAc, eluted proteins were separated by SDS-PAGE, and blots were probed with WFA or mAb IIH6. Equal amounts of cell protein from lysates of wild type ( wt ) ( B ) or mdx myotubes ( C ) treated with lobeline or control were precipitated with WFA beads, bound proteins were eluted with GalNAc, eluted proteins were separated by SDS-PAGE, and blots were probed with WFA or mAbs IIH6 or MANDAG2 ( MA ), which recognizes β-DG. Results are representative of four independent experiments.

    Article Snippet: Wild type and mdx muscle cell eluates were separated on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Millipore).

    Techniques: Binding Assay, SDS Page

    Detection of Apo-IV in chicken and quail plasma. Plasma lipoproteins with densities of ≤1.063 g/ml were delipidated, and 50 μg apolipoproteins from laying hen (lanes 1, 6, 11) or rooster (lanes 2, 7, 12), or 1 μl of whole plasma from laying hen (lanes 3, 8, 13), rooster (lanes 4, 9, 14), and quail rooster (lanes 5, 10, 15) were separated under reducing conditions on 12% SDS-polyacrylamide gels. After transfer to membranes, the blots in lanes 1–10 were incubated with rabbit anti-GST-Apo-IV antiserum (dilution 1:250) in the absence (lanes 1–5) or presence (lanes 6–10) of 10 μg/ml GST-Apo-IV, followed by peroxidase-conjugated goat anti-rabbit IgG. In lanes 11–15, preimmune rabbit serum was used instead of the anti-GST-Apo-IV rabbit antiserum.

    Journal: Biochimie

    Article Title: A novel estrogen-regulated avian apolipoprotein

    doi: 10.1016/j.biochi.2013.09.005

    Figure Lengend Snippet: Detection of Apo-IV in chicken and quail plasma. Plasma lipoproteins with densities of ≤1.063 g/ml were delipidated, and 50 μg apolipoproteins from laying hen (lanes 1, 6, 11) or rooster (lanes 2, 7, 12), or 1 μl of whole plasma from laying hen (lanes 3, 8, 13), rooster (lanes 4, 9, 14), and quail rooster (lanes 5, 10, 15) were separated under reducing conditions on 12% SDS-polyacrylamide gels. After transfer to membranes, the blots in lanes 1–10 were incubated with rabbit anti-GST-Apo-IV antiserum (dilution 1:250) in the absence (lanes 1–5) or presence (lanes 6–10) of 10 μg/ml GST-Apo-IV, followed by peroxidase-conjugated goat anti-rabbit IgG. In lanes 11–15, preimmune rabbit serum was used instead of the anti-GST-Apo-IV rabbit antiserum.

    Article Snippet: 2.4 Microsequencing The lipoprotein fraction of d ≤ 1.063 of rooster plasma was isolated by ultracentrifugal floatation, delipidated, the residue subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto a polyvinylidene difluoride membrane (Immobilon P, 0.45 mm, Millipore Corp., Bedford, MA).

    Techniques: Incubation

    Yolk VLDL does not harbor detectable amounts of Apo-IV. One μl of plasma from laying hen (LH) or rooster, or 50 μg delipidated VLDL isolated from egg yolk (yVLDL) were separated by 12% (A) or 15% (B) SDS-PAGE in the presence of 50 mM DTT. (A) Western blotting analysis of Apo-IV using mouse anti-GST-Apo-IV antiserum (dilution 1:1500), followed by peroxidase-conjugated rabbit anti-mouse IgG. (B) ApoVLDL-II was visualized by Western blot analysis using rabbit anti-apoVLDL-II antiserum (dilution 1:1500) and peroxidase-conjugated goat anti-rabbit IgG.

    Journal: Biochimie

    Article Title: A novel estrogen-regulated avian apolipoprotein

    doi: 10.1016/j.biochi.2013.09.005

    Figure Lengend Snippet: Yolk VLDL does not harbor detectable amounts of Apo-IV. One μl of plasma from laying hen (LH) or rooster, or 50 μg delipidated VLDL isolated from egg yolk (yVLDL) were separated by 12% (A) or 15% (B) SDS-PAGE in the presence of 50 mM DTT. (A) Western blotting analysis of Apo-IV using mouse anti-GST-Apo-IV antiserum (dilution 1:1500), followed by peroxidase-conjugated rabbit anti-mouse IgG. (B) ApoVLDL-II was visualized by Western blot analysis using rabbit anti-apoVLDL-II antiserum (dilution 1:1500) and peroxidase-conjugated goat anti-rabbit IgG.

    Article Snippet: 2.4 Microsequencing The lipoprotein fraction of d ≤ 1.063 of rooster plasma was isolated by ultracentrifugal floatation, delipidated, the residue subjected to SDS-polyacrylamide gel electrophoresis, and blotted onto a polyvinylidene difluoride membrane (Immobilon P, 0.45 mm, Millipore Corp., Bedford, MA).

    Techniques: Isolation, SDS Page, Western Blot

    Secreted APOL1 does not contribute to cytotoxicity. A : APOL1-myc splice variant proteins are released into culture media of transfected HEK293T cells. At 5 h after transfection, cells were transferred into DMEM supplemented with 2% FCS. After overnight incubation, culture media were collected, filtered, and precleared with Protein A/G PLUS-agarose, and APOL1-myc was immunoprecipitated using anti-myc antibodies conjugated with agarose beads. Cell lysate proteins and immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting for expression of APOL1 and GAPDH. In addition to APOL1 proteins, nonspecific proteins (most likely derived from serum-containing media) were also detected in anti-myc immunoprecipitates (asterisk). B : intracellular APOL1 is refractory to extraction with the nonionic detergent saponin. Permeabilization of cell membranes with saponin depleted cytosolic GAPDH but retained actin. At 24 h after transfection, cells were permeabilized with 0.1% saponin for 10 min, and protein lysates from untreated (Control) or saponin-treated cells (+Saponin) were analyzed by immunoblotting for expression of APOL1, GAPDH, and actin. C : secreted APOL1 does not contribute to the intracellular pool of APOL1. Transfected cells were cultured in the presence of 80 μM dynasore or solvent (DMSO). After 24 h, cells were permeabilized with 0.1% saponin, and cell protein lysates were separated by SDS-PAGE and analyzed for expression of APOL1, GAPDH, and actin. D : secreted APOL1 does not contribute to cytotoxicity. Cells were transfected with cytotoxic and noncytotoxic APOL1 variants B1 and B3, respectively, and cultured in the absence or presence of 80 μM dynasore. After 24 h, cell culture media were collected and analyzed for released LDH ( top ). Values are means ± SD of 3 independent samples. Corresponding protein lysates were analyzed by immunoblotting for expression of APOL1 and actin ( bottom ). * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Exon 4-encoded sequence is a major determinant of cytotoxicity of apolipoprotein L1

    doi: 10.1152/ajpcell.00384.2014

    Figure Lengend Snippet: Secreted APOL1 does not contribute to cytotoxicity. A : APOL1-myc splice variant proteins are released into culture media of transfected HEK293T cells. At 5 h after transfection, cells were transferred into DMEM supplemented with 2% FCS. After overnight incubation, culture media were collected, filtered, and precleared with Protein A/G PLUS-agarose, and APOL1-myc was immunoprecipitated using anti-myc antibodies conjugated with agarose beads. Cell lysate proteins and immunoprecipitated proteins were resolved by SDS-PAGE and analyzed by immunoblotting for expression of APOL1 and GAPDH. In addition to APOL1 proteins, nonspecific proteins (most likely derived from serum-containing media) were also detected in anti-myc immunoprecipitates (asterisk). B : intracellular APOL1 is refractory to extraction with the nonionic detergent saponin. Permeabilization of cell membranes with saponin depleted cytosolic GAPDH but retained actin. At 24 h after transfection, cells were permeabilized with 0.1% saponin for 10 min, and protein lysates from untreated (Control) or saponin-treated cells (+Saponin) were analyzed by immunoblotting for expression of APOL1, GAPDH, and actin. C : secreted APOL1 does not contribute to the intracellular pool of APOL1. Transfected cells were cultured in the presence of 80 μM dynasore or solvent (DMSO). After 24 h, cells were permeabilized with 0.1% saponin, and cell protein lysates were separated by SDS-PAGE and analyzed for expression of APOL1, GAPDH, and actin. D : secreted APOL1 does not contribute to cytotoxicity. Cells were transfected with cytotoxic and noncytotoxic APOL1 variants B1 and B3, respectively, and cultured in the absence or presence of 80 μM dynasore. After 24 h, cell culture media were collected and analyzed for released LDH ( top ). Values are means ± SD of 3 independent samples. Corresponding protein lysates were analyzed by immunoblotting for expression of APOL1 and actin ( bottom ). * P

    Article Snippet: After 24 h, the cells were transfected with pcDNA3.1 or APOL1 expression vector , and after an additional 24 h, cells were lysed in RIPA buffer (Santa Cruz Biotechnology), and an equal amount of lysate proteins (20–50 μg) was separated on 10% SDS-polyacrylamide gel and analyzed by immunoblotting for expression of APOL1 (HPA018885 antibody, Sigma-Aldrich), Atg5 (Cell Signaling), and actin (Sigma-Aldrich).

    Techniques: Variant Assay, Transfection, Incubation, Immunoprecipitation, SDS Page, Expressing, Derivative Assay, Cell Culture

    Expression and post-translational processing of NHE6 ΔES mutant protein is impaired in transfected AP-1 and SH-SY5Y cells. a Schematic drawing of the predicted membrane topology of mammalian NHE6v1 (based on sequence alignment and transmembrane organization of NHE1 proposed by Wakabayashi et al. [ 127 ] and Nygaard et al. [ 128 ]) and locations of mutations ( red shading ) identified by Gilfillan et al. [ 8 ] in patients with Christianson syndrome. The blue shading in the second extracellular loop (EL2) represents the additional 32 amino acids (residues 145–176) present in the NHE6v1 splice-variant. Two predicted N -glycosylation sites within EL2 are also illustrated. b AP-1 and c , SH-SY5Y cells were transiently transfected (24 h) with NHE6 HA WT or ΔES mutant. Total cell lysates of WT and ΔES-transfectants were examined by SDS-PAGE. The immunoblots were probed with a mouse monoclonal anti-HA antibody (α-HA m ) to detect NHE6v1. NHE6v1 migrates as multiple bands: slower migrating high molecular weight bands representing the fully-glycosylated ( fg ) and core-glycosylated ( cg ) dimeric forms of the exchanger (~200 and 175 kDa, respectively) that do not fully dissociate under SDS-PAGE conditions and faster migrating fully-glycosylated ( fg , ~100 kDa) and core-glycosylated ( cg , ~70 kDa) and unglycosylated ( ug , ~65 kDa) forms of the monomeric protein. To control for protein loading, the blots were reprobed with a mouse monoclonal anti-GAPDH antibody (α-GAPDH m ). d , e To confirm the nature of the oligosaccharide modifications of the NHE6 bands, AP-1 cells transiently transfected (24 h) with WT or ΔES constructs were lysed in non-detergent buffers and post-nuclear supernatants were left untreated or incubated with either endoglycosidase H (EndoH), which cleaves only asparagine-linked mannose-rich oligosaccharides (i.e., core-glycosylated) but not more highly processed complex oligosaccharides (i.e., fully-glycosylated), or peptide-N-glycosidase F (PNGaseF) which cleaves between the innermost N-acetylglucosamine and asparagine residues of all oligosaccharide structures (i.e., high mannose, hybrid, and complex). The lysates were then subjected to SDS-PAGE and immunoblotting with an α-HA m antibody. The data are representative of three independent experiments

    Journal: Molecular Neurodegeneration

    Article Title: A Christianson syndrome-linked deletion mutation (∆287ES288) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

    doi: 10.1186/s13024-016-0129-9

    Figure Lengend Snippet: Expression and post-translational processing of NHE6 ΔES mutant protein is impaired in transfected AP-1 and SH-SY5Y cells. a Schematic drawing of the predicted membrane topology of mammalian NHE6v1 (based on sequence alignment and transmembrane organization of NHE1 proposed by Wakabayashi et al. [ 127 ] and Nygaard et al. [ 128 ]) and locations of mutations ( red shading ) identified by Gilfillan et al. [ 8 ] in patients with Christianson syndrome. The blue shading in the second extracellular loop (EL2) represents the additional 32 amino acids (residues 145–176) present in the NHE6v1 splice-variant. Two predicted N -glycosylation sites within EL2 are also illustrated. b AP-1 and c , SH-SY5Y cells were transiently transfected (24 h) with NHE6 HA WT or ΔES mutant. Total cell lysates of WT and ΔES-transfectants were examined by SDS-PAGE. The immunoblots were probed with a mouse monoclonal anti-HA antibody (α-HA m ) to detect NHE6v1. NHE6v1 migrates as multiple bands: slower migrating high molecular weight bands representing the fully-glycosylated ( fg ) and core-glycosylated ( cg ) dimeric forms of the exchanger (~200 and 175 kDa, respectively) that do not fully dissociate under SDS-PAGE conditions and faster migrating fully-glycosylated ( fg , ~100 kDa) and core-glycosylated ( cg , ~70 kDa) and unglycosylated ( ug , ~65 kDa) forms of the monomeric protein. To control for protein loading, the blots were reprobed with a mouse monoclonal anti-GAPDH antibody (α-GAPDH m ). d , e To confirm the nature of the oligosaccharide modifications of the NHE6 bands, AP-1 cells transiently transfected (24 h) with WT or ΔES constructs were lysed in non-detergent buffers and post-nuclear supernatants were left untreated or incubated with either endoglycosidase H (EndoH), which cleaves only asparagine-linked mannose-rich oligosaccharides (i.e., core-glycosylated) but not more highly processed complex oligosaccharides (i.e., fully-glycosylated), or peptide-N-glycosidase F (PNGaseF) which cleaves between the innermost N-acetylglucosamine and asparagine residues of all oligosaccharide structures (i.e., high mannose, hybrid, and complex). The lysates were then subjected to SDS-PAGE and immunoblotting with an α-HA m antibody. The data are representative of three independent experiments

    Article Snippet: The immunoprecipitated proteins, along with aliquots representing the total lysate were subjected to 9 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Nepean, Ontario, Canada) for immunoblotting with a mouse monoclonal anti-ubiquitin antibody (Santa Cruz Biotechnology).

    Techniques: Expressing, Mutagenesis, Transfection, Sequencing, Variant Assay, SDS Page, Western Blot, Molecular Weight, Construct, Incubation

    Biosynthetic maturation of NHE6 is reduced for the ∆ES mutant. AP-1 cells were transiently transfected with a NHE6v1 HA WT or b ΔES and lysed at the indicated time points over a 48 h period. Equal amounts of proteins were subjected to SDS-PAGE and immunoblotting with a monoclonal anti-HA antibody (α-HA). The identities of the various NHE6 bands are as described in the legend to Fig. 1 . For the ΔES immunoblot in panel B , a longer X-ray film exposure (18X) of the 36 h and 48 h time points is also shown. The same immunoblots were also probed with a monoclonal anti-β-tubulin antibody as a loading control. c-d Densitometric quantification of the relative abundances of the monomeric and dimeric forms of WT or ΔES was assessed using ImageJ software and expressed as ratios of fully glycosylated/total protein (fg/total). For quantification, multiple exposures of the immunoblots were taken to ensure the signal intensities of the bands were within the linear range of the X-ray film. Data are shown as mean ± standard error of the mean (S.E.M.) of four different experiments

    Journal: Molecular Neurodegeneration

    Article Title: A Christianson syndrome-linked deletion mutation (∆287ES288) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

    doi: 10.1186/s13024-016-0129-9

    Figure Lengend Snippet: Biosynthetic maturation of NHE6 is reduced for the ∆ES mutant. AP-1 cells were transiently transfected with a NHE6v1 HA WT or b ΔES and lysed at the indicated time points over a 48 h period. Equal amounts of proteins were subjected to SDS-PAGE and immunoblotting with a monoclonal anti-HA antibody (α-HA). The identities of the various NHE6 bands are as described in the legend to Fig. 1 . For the ΔES immunoblot in panel B , a longer X-ray film exposure (18X) of the 36 h and 48 h time points is also shown. The same immunoblots were also probed with a monoclonal anti-β-tubulin antibody as a loading control. c-d Densitometric quantification of the relative abundances of the monomeric and dimeric forms of WT or ΔES was assessed using ImageJ software and expressed as ratios of fully glycosylated/total protein (fg/total). For quantification, multiple exposures of the immunoblots were taken to ensure the signal intensities of the bands were within the linear range of the X-ray film. Data are shown as mean ± standard error of the mean (S.E.M.) of four different experiments

    Article Snippet: The immunoprecipitated proteins, along with aliquots representing the total lysate were subjected to 9 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Nepean, Ontario, Canada) for immunoblotting with a mouse monoclonal anti-ubiquitin antibody (Santa Cruz Biotechnology).

    Techniques: Mutagenesis, Transfection, SDS Page, Western Blot, Software

    Stability of NHE6 is diminished for ΔES mutant. a AP-1 cells were transiently transfected with NHE6v1 HA WT or ΔES mutant for 24 h and then treated with 150 μg/mL cycloheximide for the indicated time points, lysed and analysed by SDS-PAGE and immunoblotting with a mouse monoclonal anti-HA (α-HA m ) antibody. Equal amounts of proteins were loaded, as shown by probing the membranes with a monoclonal anti-GAPDH antibody (α-GAPDH m ). b Quantitative analysis by densitometry of NHE6v1 WT and ΔES protein abundance (normalized to GAPDH levels) as a function of time in the presence of cycloheximide. Values represent the mean ± S.E.M. of three separate experiments. c-d AP-1 cells were transiently transfected with NHE6v1 HA WT ( c ) or ΔES ( d ) for 24 h and then treated with 150 μg/mL cycloheximide for the indicated time points in the presence of DMSO (vehicle), the proteasomal inhibitors MG-132 (40 μM) or lactacystin (LC, 30 μM) ( left panels ), or the lysosomal inhibitors leupeptin/pepstatin (LeuP, 100 μg/ml) or chloroquine (CQ, 500 μM) ( right panels ). Cellular lysates were analysed by immunoblotting with a mouse monoclonal α-HA m antibody. Membranes were also probed with a mouse monoclonal α-GAPDH m antibody as a loading control

    Journal: Molecular Neurodegeneration

    Article Title: A Christianson syndrome-linked deletion mutation (∆287ES288) in SLC9A6 disrupts recycling endosomal function and elicits neurodegeneration and cell death

    doi: 10.1186/s13024-016-0129-9

    Figure Lengend Snippet: Stability of NHE6 is diminished for ΔES mutant. a AP-1 cells were transiently transfected with NHE6v1 HA WT or ΔES mutant for 24 h and then treated with 150 μg/mL cycloheximide for the indicated time points, lysed and analysed by SDS-PAGE and immunoblotting with a mouse monoclonal anti-HA (α-HA m ) antibody. Equal amounts of proteins were loaded, as shown by probing the membranes with a monoclonal anti-GAPDH antibody (α-GAPDH m ). b Quantitative analysis by densitometry of NHE6v1 WT and ΔES protein abundance (normalized to GAPDH levels) as a function of time in the presence of cycloheximide. Values represent the mean ± S.E.M. of three separate experiments. c-d AP-1 cells were transiently transfected with NHE6v1 HA WT ( c ) or ΔES ( d ) for 24 h and then treated with 150 μg/mL cycloheximide for the indicated time points in the presence of DMSO (vehicle), the proteasomal inhibitors MG-132 (40 μM) or lactacystin (LC, 30 μM) ( left panels ), or the lysosomal inhibitors leupeptin/pepstatin (LeuP, 100 μg/ml) or chloroquine (CQ, 500 μM) ( right panels ). Cellular lysates were analysed by immunoblotting with a mouse monoclonal α-HA m antibody. Membranes were also probed with a mouse monoclonal α-GAPDH m antibody as a loading control

    Article Snippet: The immunoprecipitated proteins, along with aliquots representing the total lysate were subjected to 9 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Nepean, Ontario, Canada) for immunoblotting with a mouse monoclonal anti-ubiquitin antibody (Santa Cruz Biotechnology).

    Techniques: Mutagenesis, Transfection, SDS Page

    Effect of compounds 1 , 2 and sorafenib (as a control) on the level of expression of phosphorylated (activated) and total ERK. HL-60 and MCF-7 cells were treated with either vehicle (0.1% EtOH), sorafenib (10 μM), compound 1 (10 μM) or compound 2 (10 μM) for 16 hours. Cells were lysed and 20 μg protein was loaded, separated on 12% SDS-page gel and transferred to PVDF membrane. The membrane was then blocked and probed with anti-ERK and anti-pERK [1 : 1000] and anti-rabbit secondary antibodies. Anti-GAPDH [1 : 2500] was used as a loading control and probed with anti-mouse secondary. HL-60 and MCF-7 results are representative of 4 and 2 independent experiments respectively.

    Journal: MedChemComm

    Article Title: Effect of isouronium/guanidinium substitution on the efficacy of a series of novel anti-cancer agents of isouronium/guanidinium substitution on the efficacy of a series of novel anti-cancer agents †Electronic supplementary information (ESI) available: Computational results, synthetic general procedures and preparation of intermediates, NMR spectra and HPLC chromatogram of final compounds and calculated physicochemical properties. See DOI: 10.1039/c8md00089a

    doi: 10.1039/c8md00089a

    Figure Lengend Snippet: Effect of compounds 1 , 2 and sorafenib (as a control) on the level of expression of phosphorylated (activated) and total ERK. HL-60 and MCF-7 cells were treated with either vehicle (0.1% EtOH), sorafenib (10 μM), compound 1 (10 μM) or compound 2 (10 μM) for 16 hours. Cells were lysed and 20 μg protein was loaded, separated on 12% SDS-page gel and transferred to PVDF membrane. The membrane was then blocked and probed with anti-ERK and anti-pERK [1 : 1000] and anti-rabbit secondary antibodies. Anti-GAPDH [1 : 2500] was used as a loading control and probed with anti-mouse secondary. HL-60 and MCF-7 results are representative of 4 and 2 independent experiments respectively.

    Article Snippet: Equal protein concentrations of lysates were resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF transfer membrane (EMD Millipore, Germany).

    Techniques: Expressing, SDS Page

    Gastrin induced phosphorylation of protein kinase C (PKC)-δ. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, 30, or 60 minutes and cell lysates were then obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to membranes. Membranes were reacted with rabbit antihuman-phospho-PKC-δ (Thr505) polyclonal antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system.

    Journal: Gut

    Article Title: Gastrin activates nuclear factor ?B (NF?B) through a protein kinase C dependent pathway involving NF?B inducing kinase, inhibitor ?B (I?B) kinase, and tumour necrosis factor receptor associated factor 6 (TRAF6) in MKN-28 cells transfected with gastrin receptor

    doi:

    Figure Lengend Snippet: Gastrin induced phosphorylation of protein kinase C (PKC)-δ. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, 30, or 60 minutes and cell lysates were then obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to membranes. Membranes were reacted with rabbit antihuman-phospho-PKC-δ (Thr505) polyclonal antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system.

    Article Snippet: Aliquots containing 50 μg of total protein were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (5–20% gradient gels), and proteins were transferred to polyvinylidine difluoride membranes (Immobilon; Millipore, Bedford, Massachusetts, USA).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation

    Gastrin induced degradation of inhibitor κB (IκB)-α. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, or 30 minutes and cell lysates were obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidine difluoride membranes. Membranes were reacted with rabbit antihuman-IκB-α antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system. Quantitated signals were determined by densitometry.

    Journal: Gut

    Article Title: Gastrin activates nuclear factor ?B (NF?B) through a protein kinase C dependent pathway involving NF?B inducing kinase, inhibitor ?B (I?B) kinase, and tumour necrosis factor receptor associated factor 6 (TRAF6) in MKN-28 cells transfected with gastrin receptor

    doi:

    Figure Lengend Snippet: Gastrin induced degradation of inhibitor κB (IκB)-α. MKGR26 cells and MKN-neo cells were treated with gastrin for 0, 5, 15, or 30 minutes and cell lysates were obtained. Proteins were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and then transferred to polyvinylidine difluoride membranes. Membranes were reacted with rabbit antihuman-IκB-α antibody, incubated with peroxidase conjugated goat antirabbit IgG, and peroxidase was detected with an enhanced chemiluminescence system. Quantitated signals were determined by densitometry.

    Article Snippet: Aliquots containing 50 μg of total protein were size fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (5–20% gradient gels), and proteins were transferred to polyvinylidine difluoride membranes (Immobilon; Millipore, Bedford, Massachusetts, USA).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation

    Identification of a YlxM and Ffh interaction. (A) ELISAs showing immobilized YlxM (or MBP as a negative control) incubated with serial dilutions of Ffh. Bound protein was detected with monospecific polyclonal rabbit anti-Ffh antisera. (B) Coomassie blue stain of purified recombinant YlxM (rYlxM) that was covalently coupled to Sepharose to generate the affinity matrix. (C) Western blot analysis of S. mutans whole-cell lysates applied to the YlxM affinity matrix (Beads with YlxM) or a bead-only control. Captured proteins were eluted as described in Materials and Methods and subjected to SDS-polyacrylamide gel electrophoresis, electroblotted onto a PVDF membrane, and reacted with polyclonal rabbit anti-Ffh, -FtsY, -YlxM, or -6His antibodies.

    Journal: Journal of Bacteriology

    Article Title: YlxM Is a Newly Identified Accessory Protein That Influences the Function of Signal Recognition Particle Pathway Components in Streptococcus mutans

    doi: 10.1128/JB.01465-13

    Figure Lengend Snippet: Identification of a YlxM and Ffh interaction. (A) ELISAs showing immobilized YlxM (or MBP as a negative control) incubated with serial dilutions of Ffh. Bound protein was detected with monospecific polyclonal rabbit anti-Ffh antisera. (B) Coomassie blue stain of purified recombinant YlxM (rYlxM) that was covalently coupled to Sepharose to generate the affinity matrix. (C) Western blot analysis of S. mutans whole-cell lysates applied to the YlxM affinity matrix (Beads with YlxM) or a bead-only control. Captured proteins were eluted as described in Materials and Methods and subjected to SDS-polyacrylamide gel electrophoresis, electroblotted onto a PVDF membrane, and reacted with polyclonal rabbit anti-Ffh, -FtsY, -YlxM, or -6His antibodies.

    Article Snippet: Cell lysates were electrophoresed through 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels, transblotted onto Immobilon polyvinylidene difluoride (PVDF) membranes (Sigma-Aldrich, St. Louis, MO), reacted with anti-YlxM affinity-purified antibodies followed by horseradish peroxidase-labeled anti-rabbit IgG, and developed using the enhanced-chemiluminescence (ECL) Western blotting system (GE Healthcare) and Hyperfilm (GE Healthcare) according to the manufacturer's instructions.

    Techniques: Negative Control, Incubation, Staining, Purification, Recombinant, Western Blot, Polyacrylamide Gel Electrophoresis

    Arginines (R44, R53 and R57) in the recognition helix and N-terminal arm (R3 and R5) of the homeodomain of Pax6 are important for the interaction with the paired domain. GST pull-down assays with Pax6 HD, and HD mutations fused to GST and immobilized on glutathione–agarose beads and in vitro translated Pax6ΔHD. ( A ) The GST pull-downs were performed as described in the legend to Figure 1 . The panel shows two different experiments using two different GST-HD fusions. The GST-HD protein (upper panel) contains two amino acids N-terminal to the HD while the GST-18L-HD (lower panel) contains 18 amino acids of the linker region N-terminal to the HD to study the effect of mutating also at −3 relative to the start of the HD. ( B ) Quantitative representation of the interaction data. A Fuji Bio-imaging analyzer (BAS5000) equipped with Image Gauge version 4.0 software was used to quantitate 35 S-labeled proteins in the SDS–polyacrylamide gels. The amount 35 S-labeled Pax6ΔHD pulled down by wild-type GST-HD was set to 100%. The data shown represent the mean of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions

    doi: 10.1093/nar/gki562

    Figure Lengend Snippet: Arginines (R44, R53 and R57) in the recognition helix and N-terminal arm (R3 and R5) of the homeodomain of Pax6 are important for the interaction with the paired domain. GST pull-down assays with Pax6 HD, and HD mutations fused to GST and immobilized on glutathione–agarose beads and in vitro translated Pax6ΔHD. ( A ) The GST pull-downs were performed as described in the legend to Figure 1 . The panel shows two different experiments using two different GST-HD fusions. The GST-HD protein (upper panel) contains two amino acids N-terminal to the HD while the GST-18L-HD (lower panel) contains 18 amino acids of the linker region N-terminal to the HD to study the effect of mutating also at −3 relative to the start of the HD. ( B ) Quantitative representation of the interaction data. A Fuji Bio-imaging analyzer (BAS5000) equipped with Image Gauge version 4.0 software was used to quantitate 35 S-labeled proteins in the SDS–polyacrylamide gels. The amount 35 S-labeled Pax6ΔHD pulled down by wild-type GST-HD was set to 100%. The data shown represent the mean of three independent experiments.

    Article Snippet: The GST fusion proteins were analyzed on a 10% SDS–polyacrylamide gel and quantified using SYPRO® Ruby protein stain (Bio-Rad) and LumiAnalyst imager and software (Roche Applied Sciences).

    Techniques: In Vitro, Imaging, Software, Labeling

    The recognition helix of the homeodomain of Pax6 is important for interaction with both the PD and the HD. ( A ) Pax6 constructs used for in vitro translation and GST pull-downs. ( B ) GST pull-down assays with Pax6 HD and PD fused to GST and immobilized on glutathione–agarose beads and Pax6ΔPDΔHD, Pax6ΔPDΔh2–3 or Pax6ΔPDΔh3 produced by in vitro transcription and translation in the presence of [ 35 S]methionine. An aliquot of 10 μl of the in vitro translation reactions was preincubated with GST immobilized on glutathione–agarose beads before incubation with the GST fusion proteins. The GST beads, GST-Pax6 HD beads and GST-Pax6 PD beads were washed several times before they were boiled in SDS loading buffer and run on a 10% SDS–polyacrylamide gel. An aliquot of 2 μl of the in vitro translated proteins was run on the same gel to visualize the signal from 20% of the input. ( C ) Point mutations in helix 3 of the homeodomain strongly reduce the ability of Pax6 HD to interact with the PD and the wild-type HD. The N51Q, R53A and R58A, but not S50A, mutants impede the HD–PD and HD–HD interactions. GST pull-down assays were performed with recombinant GST fusions of wild type or mutants of Pax6ΔHD against in vitro translated, [ 35 S]methionine-labeled Pax6, Pax6ΔHD or Pax6ΔPD. ( D ) The interactions between full-length Pax6 and the RED subdomain and between full-length Pax6 and the HD are independent of DNA. GST pull-down assays were done with Pax6 HD and RED fused to GST as in (C). Where indicated, the pull-down experiments were performed in the presence of 500 U benzonase to degrade both DNA and RNA. The results shown are representative of three independent experiments. ( E ) The PD–HD and HD–HD interactions of Pax6 are also observed in the yeast-based SOS recruitment interaction system. The temperature sensitive yeast strain S.cerevisiae cdc25-2 MATa was co-transformed either with pSOS-zfPax6-HDwt and empty pMYR or pMYR-LaminC as negative controls, pMYR-SOS binding protein as a positive control, pMYR-zfPax6-HDwt, or with pMYR-zfPax6-PDwt. Three independent colonies generated from each co-transformation were replica plated onto galactose plates and grown in parallel at 25 and 37°C for 6 days. The results shown are representative of three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: The third helix of the homeodomain of paired class homeodomain proteins acts as a recognition helix both for DNA and protein interactions

    doi: 10.1093/nar/gki562

    Figure Lengend Snippet: The recognition helix of the homeodomain of Pax6 is important for interaction with both the PD and the HD. ( A ) Pax6 constructs used for in vitro translation and GST pull-downs. ( B ) GST pull-down assays with Pax6 HD and PD fused to GST and immobilized on glutathione–agarose beads and Pax6ΔPDΔHD, Pax6ΔPDΔh2–3 or Pax6ΔPDΔh3 produced by in vitro transcription and translation in the presence of [ 35 S]methionine. An aliquot of 10 μl of the in vitro translation reactions was preincubated with GST immobilized on glutathione–agarose beads before incubation with the GST fusion proteins. The GST beads, GST-Pax6 HD beads and GST-Pax6 PD beads were washed several times before they were boiled in SDS loading buffer and run on a 10% SDS–polyacrylamide gel. An aliquot of 2 μl of the in vitro translated proteins was run on the same gel to visualize the signal from 20% of the input. ( C ) Point mutations in helix 3 of the homeodomain strongly reduce the ability of Pax6 HD to interact with the PD and the wild-type HD. The N51Q, R53A and R58A, but not S50A, mutants impede the HD–PD and HD–HD interactions. GST pull-down assays were performed with recombinant GST fusions of wild type or mutants of Pax6ΔHD against in vitro translated, [ 35 S]methionine-labeled Pax6, Pax6ΔHD or Pax6ΔPD. ( D ) The interactions between full-length Pax6 and the RED subdomain and between full-length Pax6 and the HD are independent of DNA. GST pull-down assays were done with Pax6 HD and RED fused to GST as in (C). Where indicated, the pull-down experiments were performed in the presence of 500 U benzonase to degrade both DNA and RNA. The results shown are representative of three independent experiments. ( E ) The PD–HD and HD–HD interactions of Pax6 are also observed in the yeast-based SOS recruitment interaction system. The temperature sensitive yeast strain S.cerevisiae cdc25-2 MATa was co-transformed either with pSOS-zfPax6-HDwt and empty pMYR or pMYR-LaminC as negative controls, pMYR-SOS binding protein as a positive control, pMYR-zfPax6-HDwt, or with pMYR-zfPax6-PDwt. Three independent colonies generated from each co-transformation were replica plated onto galactose plates and grown in parallel at 25 and 37°C for 6 days. The results shown are representative of three independent experiments.

    Article Snippet: The GST fusion proteins were analyzed on a 10% SDS–polyacrylamide gel and quantified using SYPRO® Ruby protein stain (Bio-Rad) and LumiAnalyst imager and software (Roche Applied Sciences).

    Techniques: Construct, In Vitro, Produced, Incubation, Recombinant, Labeling, Transformation Assay, Binding Assay, Positive Control, Generated

    Production of Nb displaying LVs. ( a ) Non-modified HEK 293T cells or HEK 293T cells stably expressing Nb BCII10 or DC2.1 were used to produce LVs pseudotyped with VSV.G or VSV.GS, respectively. Three days after transfection of these cells with the VSV.G or VSV.GS, gag/pol and transgene encoding plasmids, we evaluated the expression of the transgene (Thy1.1 or tNGFR), as well as the Nb (myc tag) by flow cytometry. Non-transfected HEK 293T cells served as a control. The flow cytometry dot plots demonstrate high expression of the transgene ( y axis) in all transfected cells (VSV.G, BCII10 and DC2.1) and high expression of Nbs ( x -axis) on the Nb-modified HEK 293T cells (BCII10 and DC2.1). One representative experiment is shown ( n =6). ( b ) To compare the LV preparations we determined their RT content. The graph depicts the amount of RT (ng RT/μl) in the LV preparations. Each dot represents one LV stock, the horizontal line shows the mean ( n =6). ( c ) An ELISA involving anti-VHH and anti-Thy1.1 as capture and detection antibodies, respectively, was used to demonstrate the incorporation of Nbs into the surface of Thy1.1 encoding LVs. A serial dilution of LVs was applied (5, 2.5 and 1.25 ng RT). The graph depicts the OD-values detected. One representative experiment is shown ( n =3). ( d ) Western blot was performed as a quality control of the LVs. After separation on a 15% sodium dodecyl sulphate-polyacrylamide gel and transfer to a nitrocellulose membrane, the Nbs (±25 kDa) and VSV.GS (±15 kDa), which both contain an HA epitope tag, were detected with an anti-HA antibody. One representative experiment is shown ( n =4). ( e ) The density of the western blot signals was determined using the Photocapt MW software and used to determine the ratio of Nbs/VSV.GS on the LVs. This ratio is shown in the graph, in which each dot represents one LV stock and the horizontal line shows the mean ( n =4).

    Journal: Gene Therapy

    Article Title: Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells

    doi: 10.1038/gt.2011.206

    Figure Lengend Snippet: Production of Nb displaying LVs. ( a ) Non-modified HEK 293T cells or HEK 293T cells stably expressing Nb BCII10 or DC2.1 were used to produce LVs pseudotyped with VSV.G or VSV.GS, respectively. Three days after transfection of these cells with the VSV.G or VSV.GS, gag/pol and transgene encoding plasmids, we evaluated the expression of the transgene (Thy1.1 or tNGFR), as well as the Nb (myc tag) by flow cytometry. Non-transfected HEK 293T cells served as a control. The flow cytometry dot plots demonstrate high expression of the transgene ( y axis) in all transfected cells (VSV.G, BCII10 and DC2.1) and high expression of Nbs ( x -axis) on the Nb-modified HEK 293T cells (BCII10 and DC2.1). One representative experiment is shown ( n =6). ( b ) To compare the LV preparations we determined their RT content. The graph depicts the amount of RT (ng RT/μl) in the LV preparations. Each dot represents one LV stock, the horizontal line shows the mean ( n =6). ( c ) An ELISA involving anti-VHH and anti-Thy1.1 as capture and detection antibodies, respectively, was used to demonstrate the incorporation of Nbs into the surface of Thy1.1 encoding LVs. A serial dilution of LVs was applied (5, 2.5 and 1.25 ng RT). The graph depicts the OD-values detected. One representative experiment is shown ( n =3). ( d ) Western blot was performed as a quality control of the LVs. After separation on a 15% sodium dodecyl sulphate-polyacrylamide gel and transfer to a nitrocellulose membrane, the Nbs (±25 kDa) and VSV.GS (±15 kDa), which both contain an HA epitope tag, were detected with an anti-HA antibody. One representative experiment is shown ( n =4). ( e ) The density of the western blot signals was determined using the Photocapt MW software and used to determine the ratio of Nbs/VSV.GS on the LVs. This ratio is shown in the graph, in which each dot represents one LV stock and the horizontal line shows the mean ( n =4).

    Article Snippet: Viral proteins were separated on a 15% sodium dodecyl sulfate-polyacrylamide gel, transferred to a nitrocellulose membrane, after which Nbs and VSV.GS, which contain an HA tag, were detected with an anti-HA antibody (Sigma-Aldrich) and a horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Santa Cruz Biotechnology, Heidelberg, Germany) as primary and secondary antibody, respectively.

    Techniques: Modification, Stable Transfection, Expressing, Transfection, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Serial Dilution, Western Blot, Software

    Identification of NFM as the target antigen of non-NMDAR autoantibodies. We cotransfected HEK293T cells with GFP and NFM, NFH, NFL, or βIII-tubulin (A, column 1); column 2 shows the immunostaining for a commercially available antibody in red, and column 3 shows the merged reactivities. The non-NMDAR autoantibodies in the serum from patients reacted with HEK293T cells expressing human NFM in a fluorescence-based cell-binding assay (B). From top to bottom: binding of antibodies from patients' serum or CSF using an antihuman immunoglobulin G secondary antibody (red) to cells transfected with human NFM (green), merged images (yellow), and the absence of staining of nontransfected cells. Patients with anti-NMDAR encephalitis were negative for NFH, NFL, and βIII-tubulin antibodies, while cells displayed filamentous patterns. No binding was observed when using healthy serum. Representative results obtained by Western blotting (“P” and “C” indicate specific patients and controls, respectively) (C). Whole-cell lysates prepared from human HEK293T cells with (+) or without (−) transfection of the human NFM gene were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The blots were exposed to serum from seven patients with anti-NMDAR encephalitis and seven healthy controls (C1–C3) after a total of 20 µg of protein lysates from HEK293T cells. Antibodies that bound to the HEK293T cell lysates transfected with NFM that predominantly reacted with one antigen of approximately 160 kDa were seen in seru m (diluted 1:100) from patients 3, 13, 24, 39, 43, 46, and 51. This binding was not observed in controls. No protein bands against the HEK293T cells without transfection (as negative controls) were detected in any of the samples. NFM was detected using commercially available anti-NFM antibodies as a positive control. Scale bar=20 µm. CSF: cerebrospinal fluid, GFP: green fluorescent protein, HEK: human embryonic kidney, NFH: heavy neurofilament, NFL: light neurofilament, NFM: medium neurofilament, NMDAR: N-methyl-D-aspartate receptor.

    Journal: Journal of Clinical Neurology (Seoul, Korea)

    Article Title: Identification of Medium-Length Antineurofilament Autoantibodies in Patients with Anti-N-Methyl-D-Aspartate Receptor Encephalitis

    doi: 10.3988/jcn.2020.16.3.470

    Figure Lengend Snippet: Identification of NFM as the target antigen of non-NMDAR autoantibodies. We cotransfected HEK293T cells with GFP and NFM, NFH, NFL, or βIII-tubulin (A, column 1); column 2 shows the immunostaining for a commercially available antibody in red, and column 3 shows the merged reactivities. The non-NMDAR autoantibodies in the serum from patients reacted with HEK293T cells expressing human NFM in a fluorescence-based cell-binding assay (B). From top to bottom: binding of antibodies from patients' serum or CSF using an antihuman immunoglobulin G secondary antibody (red) to cells transfected with human NFM (green), merged images (yellow), and the absence of staining of nontransfected cells. Patients with anti-NMDAR encephalitis were negative for NFH, NFL, and βIII-tubulin antibodies, while cells displayed filamentous patterns. No binding was observed when using healthy serum. Representative results obtained by Western blotting (“P” and “C” indicate specific patients and controls, respectively) (C). Whole-cell lysates prepared from human HEK293T cells with (+) or without (−) transfection of the human NFM gene were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The blots were exposed to serum from seven patients with anti-NMDAR encephalitis and seven healthy controls (C1–C3) after a total of 20 µg of protein lysates from HEK293T cells. Antibodies that bound to the HEK293T cell lysates transfected with NFM that predominantly reacted with one antigen of approximately 160 kDa were seen in seru m (diluted 1:100) from patients 3, 13, 24, 39, 43, 46, and 51. This binding was not observed in controls. No protein bands against the HEK293T cells without transfection (as negative controls) were detected in any of the samples. NFM was detected using commercially available anti-NFM antibodies as a positive control. Scale bar=20 µm. CSF: cerebrospinal fluid, GFP: green fluorescent protein, HEK: human embryonic kidney, NFH: heavy neurofilament, NFL: light neurofilament, NFM: medium neurofilament, NMDAR: N-methyl-D-aspartate receptor.

    Article Snippet: The extracted protein samples were heat-denatured and then electrophoresed in an 8% sodium dodecyl sulfate–polyacrylamide gel, and the separated proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories).

    Techniques: Immunostaining, Expressing, Fluorescence, Cell Binding Assay, Binding Assay, Transfection, Staining, Western Blot, Polyacrylamide Gel Electrophoresis, Positive Control

    CPV purification and characterization. A. Sucrose gradient purification . VLPs preparation from infected cell culture lysates purified by sucrose gradient centrifugation (10–40%). Bands of CPV-VLPs that were derivatized with OG-488 are visible in the gradient just above the middle of the tube (left panel) and appear fluorescent green under a UV-light source (right panel). B. SDS-PAGE analyses . The purified VLPs were subjected to electrophoresis in 4–12% Bis-tris gel and stained with SimplyBlue (Invitrogen) to reveal the proteins (left panel). The Seeblue plus protein molecular weight standards in kDa (Invitrogen) are indicated on the side of the gel picture (lane 1). Lanes 2 and 3 contain protein from CPV-VLPs derivatized with OG-488 and CPV-VLPs respectively. Prior to staining, the gel (right panel) visualized with a UV-light source showed a fluorescent 62 kDa band in the lane of OG-488 derivatized CPV-VLPs (lane 2f) and lacked any fluorescent bands in the native CPV-VLPs (lane 3f).

    Journal: Journal of Nanobiotechnology

    Article Title: Canine parvovirus-like particles, a novel nanomaterial for tumor targeting

    doi: 10.1186/1477-3155-4-2

    Figure Lengend Snippet: CPV purification and characterization. A. Sucrose gradient purification . VLPs preparation from infected cell culture lysates purified by sucrose gradient centrifugation (10–40%). Bands of CPV-VLPs that were derivatized with OG-488 are visible in the gradient just above the middle of the tube (left panel) and appear fluorescent green under a UV-light source (right panel). B. SDS-PAGE analyses . The purified VLPs were subjected to electrophoresis in 4–12% Bis-tris gel and stained with SimplyBlue (Invitrogen) to reveal the proteins (left panel). The Seeblue plus protein molecular weight standards in kDa (Invitrogen) are indicated on the side of the gel picture (lane 1). Lanes 2 and 3 contain protein from CPV-VLPs derivatized with OG-488 and CPV-VLPs respectively. Prior to staining, the gel (right panel) visualized with a UV-light source showed a fluorescent 62 kDa band in the lane of OG-488 derivatized CPV-VLPs (lane 2f) and lacked any fluorescent bands in the native CPV-VLPs (lane 3f).

    Article Snippet: CPV-VLPs denatured in SDS-PAGE sample buffer were separated in a 4–12% bis-tris polyacrylamide gel (Invitrogen, Carlsbad, CA) by employing a 200 V constant current for 35 minutes.

    Techniques: Purification, Infection, Cell Culture, Gradient Centrifugation, SDS Page, Electrophoresis, Staining, Molecular Weight

    Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% SDS-PAGE gels and transferred to PVDF membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments

    Journal: The Journal of steroid biochemistry and molecular biology

    Article Title: Functional characterization of the G162R and D216H genetic variants of human CYP17A1

    doi: 10.1016/j.jsbmb.2017.12.002

    Figure Lengend Snippet: Polyubiquitination of G162R variant protein when expressed in HEK-293T cells HEK-293T cells were transiently transfected with either WT CYP17A1 or G162R constructs for 48 hours. For the indicated times prior to protein collection, the cells were treated with 10 μM MG132. Protein was collected using RIPA lysis buffer supplemented with a protease inhibitor tablet and 5 mM NEM to prevent deubiquitination. Whole cells lysates were subjected to immunoblot analysis on 10% SDS-PAGE gels and transferred to PVDF membrane. Blots were probed with anti CYP17A1 antibody and imaged on film using a HRP conjugated secondary antibody and chemiluminescent substrate. Blot represents 1 of 3 independent experiments

    Article Snippet: Protein (30–50 μg per lane) was loaded, resolved on a 10% SDS-polyacrylamide gel (ThermoScientific), and transferred onto a PVDF membrane.

    Techniques: Variant Assay, Transfection, Construct, Lysis, Protease Inhibitor, SDS Page

    POLA1 levels are elevated in colorectal cancer and ST1926 decreases its expression. A. ST1926 reduces POLA1 protein levels in colorectal cell lines. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to two days. Total SDS protein lysates (80 µg/lane) were immunoblotted against POLA1 antibody. Similar trends in protein levels were observed in two independent experiments. B. POLA1 basal protein levels are elevated in CRC cell lines compared to the normal-like NCM460 cells. Total SDS protein lysates (80 µg/lane) were immunoblotted against POLA1 antibody. Similar trends were observed in two independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. Expression levels of POLA1 ]. D. POLA1 ]. P values

    Journal: American Journal of Cancer Research

    Article Title: Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α

    doi:

    Figure Lengend Snippet: POLA1 levels are elevated in colorectal cancer and ST1926 decreases its expression. A. ST1926 reduces POLA1 protein levels in colorectal cell lines. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to two days. Total SDS protein lysates (80 µg/lane) were immunoblotted against POLA1 antibody. Similar trends in protein levels were observed in two independent experiments. B. POLA1 basal protein levels are elevated in CRC cell lines compared to the normal-like NCM460 cells. Total SDS protein lysates (80 µg/lane) were immunoblotted against POLA1 antibody. Similar trends were observed in two independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. Expression levels of POLA1 ]. D. POLA1 ]. P values

    Article Snippet: Total cellular proteins (50 µg) were loaded onto a 10% SDS-polyacrylamide gel, subjected to electrophoresis and immunoblotted with the following antibodies: p53 (sc-126), p21 (sc-397), and PARP (sc-7150) from Santa Cruz Biotechnology (Heidelberg, Germany), γ-H2AX (2577) from Cell Signaling, Danvers, MA, POLA1 (31777) from Abcam (Cambridge, UK), and GAPDH (MAB5476) (Abnova, Heidelberg, Germany).

    Techniques: Expressing

    ST1926-resistant cells are cross-resistant to CD437 and both compounds inhibit POLA1 activity. A. Effect of ST1926 treatment on the growth of human HCT116-STR. ST1926-resistant cells (HCT116-STR) were generated by treating the parental cell line with increasing concentrations of ST1926 over a period of eight months. HCT116-STR cells were seeded in 96-well plates at a density of 5,000 cells/well, and treated with the indicated concentrations of ST1926 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of three independent experiments ± SEM. B. HCT116-STR DNA damage response to ST1926 treatment. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to 24 hours. Total SDS protein lysates (50 µg/lane) were immunoblotted against γ-H2AX antibody. Similar trends in protein levels were observed in three independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. TUNEL analysis of HCT116-STR cells treated with 0.1% DMSO or 1 μM ST1926 up to 48 hours. Results are representative of two independent experiments. D. Effect of CD437 treatment on HCT116 and HCT116-STR cell growth. Cells were treated with the indicated concentrations of CD437 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of at least three independent experiments ± SEM. E. Primer extension assay was evaluated in the presence of increasing concentrations of ST1926 and CD437 (0.069, 0.21, 0.62, 1.9, 5.6, 17, 50, and 100 μM). “-” represents the control (DMSO). The activity of POLA1 was determined by the detection of primer of a 25-nucleotide product, and a gel representative of two independent experiments is shown.

    Journal: American Journal of Cancer Research

    Article Title: Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α

    doi:

    Figure Lengend Snippet: ST1926-resistant cells are cross-resistant to CD437 and both compounds inhibit POLA1 activity. A. Effect of ST1926 treatment on the growth of human HCT116-STR. ST1926-resistant cells (HCT116-STR) were generated by treating the parental cell line with increasing concentrations of ST1926 over a period of eight months. HCT116-STR cells were seeded in 96-well plates at a density of 5,000 cells/well, and treated with the indicated concentrations of ST1926 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of three independent experiments ± SEM. B. HCT116-STR DNA damage response to ST1926 treatment. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to 24 hours. Total SDS protein lysates (50 µg/lane) were immunoblotted against γ-H2AX antibody. Similar trends in protein levels were observed in three independent experiments. Blots were reprobed with GAPDH antibody to ensure equal protein loading. C. TUNEL analysis of HCT116-STR cells treated with 0.1% DMSO or 1 μM ST1926 up to 48 hours. Results are representative of two independent experiments. D. Effect of CD437 treatment on HCT116 and HCT116-STR cell growth. Cells were treated with the indicated concentrations of CD437 for up to three days. Cell growth was assayed in triplicate wells using the MTT assay. Results are expressed as percentage of control (0.1% DMSO), and represent the average of at least three independent experiments ± SEM. E. Primer extension assay was evaluated in the presence of increasing concentrations of ST1926 and CD437 (0.069, 0.21, 0.62, 1.9, 5.6, 17, 50, and 100 μM). “-” represents the control (DMSO). The activity of POLA1 was determined by the detection of primer of a 25-nucleotide product, and a gel representative of two independent experiments is shown.

    Article Snippet: Total cellular proteins (50 µg) were loaded onto a 10% SDS-polyacrylamide gel, subjected to electrophoresis and immunoblotted with the following antibodies: p53 (sc-126), p21 (sc-397), and PARP (sc-7150) from Santa Cruz Biotechnology (Heidelberg, Germany), γ-H2AX (2577) from Cell Signaling, Danvers, MA, POLA1 (31777) from Abcam (Cambridge, UK), and GAPDH (MAB5476) (Abnova, Heidelberg, Germany).

    Techniques: Activity Assay, Generated, MTT Assay, TUNEL Assay, Primer Extension Assay

    ST1926 treatment results in loss of mitochondrial membrane potential and early DNA damage, independently of p53 and p21. (A) Dissipation of the mitochondrial membrane potential of colorectal cancer cells by ST1926. HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells were treated with 0.1% DMSO or 1 µM ST1926 for two days, then stained with Rhodamine-123. Accumulation of Rhodamine-123 fluorescent dye was measured by flow cytometry, and is represented in histograms showing an overlay of ST1926-treated cells over control cells. Results are representative of three independent experiments and numbers indicate percentage of cells with mitochondrial dissipation. (B) ST1926 increases the protein levels of p53 and p21, and (C) γ-H2AX in HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to two days. Total SDS protein lysates (50 µg/lane) were immunoblotted against p53, p21, and γ-H2AX antibodies. Similar trends in protein levels were observed in three independent experiments. All blots were reprobed with GAPDH antibody to ensure equal protein loading (B, C).

    Journal: American Journal of Cancer Research

    Article Title: Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α

    doi:

    Figure Lengend Snippet: ST1926 treatment results in loss of mitochondrial membrane potential and early DNA damage, independently of p53 and p21. (A) Dissipation of the mitochondrial membrane potential of colorectal cancer cells by ST1926. HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells were treated with 0.1% DMSO or 1 µM ST1926 for two days, then stained with Rhodamine-123. Accumulation of Rhodamine-123 fluorescent dye was measured by flow cytometry, and is represented in histograms showing an overlay of ST1926-treated cells over control cells. Results are representative of three independent experiments and numbers indicate percentage of cells with mitochondrial dissipation. (B) ST1926 increases the protein levels of p53 and p21, and (C) γ-H2AX in HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells. Cells were treated with 0.1% DMSO or 1 μM ST1926 for up to two days. Total SDS protein lysates (50 µg/lane) were immunoblotted against p53, p21, and γ-H2AX antibodies. Similar trends in protein levels were observed in three independent experiments. All blots were reprobed with GAPDH antibody to ensure equal protein loading (B, C).

    Article Snippet: Total cellular proteins (50 µg) were loaded onto a 10% SDS-polyacrylamide gel, subjected to electrophoresis and immunoblotted with the following antibodies: p53 (sc-126), p21 (sc-397), and PARP (sc-7150) from Santa Cruz Biotechnology (Heidelberg, Germany), γ-H2AX (2577) from Cell Signaling, Danvers, MA, POLA1 (31777) from Abcam (Cambridge, UK), and GAPDH (MAB5476) (Abnova, Heidelberg, Germany).

    Techniques: Staining, Flow Cytometry, Cytometry

    ST1926 induces S-phase arrest and apoptosis in colorectal cancer cells. A. ST1926 treatment causes accumulation of cells in the sub-G 1 region. B. Cell cycle distribution of colorectal cancer cells. HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells were treated with 0.1% DMSO or 1 μM ST1926 up to two days and stained with propidium iodide (50 mg/ml). The sub-G 1 percentage presumably indicates apoptotic cells. The sum of G 0 /G 1 , S, and G 2 /M phases is a percentage of nonapoptotic cells at days one and two post-ST1926 treatment. Percentage cells in the G 0 /G 1 phase are calculated as 100 - (S+G 2 /M). Results represent the average of three independent experiments (± SEM). C. TUNEL analysis of colorectal cancer cells treated with 0.1% DMSO or 1 μM ST1926 up to two days. Histograms are representative of two independent experiments and numbers indicate percentage of TUNEL-positive cells. D. ST1926 causes PARP cleavage in colorectal cancer cells. Cells were treated with 0.1% DMSO or 1 μM ST1926 up to two days. Total SDS protein lysates (50 µg/lane) were immunoblotted against PARP antibody. Arrow indicates cleaved PARP subunit. Similar trends were observed in three independent experiments. *P

    Journal: American Journal of Cancer Research

    Article Title: Mechanism of action of the atypical retinoid ST1926 in colorectal cancer: DNA damage and DNA polymerase α

    doi:

    Figure Lengend Snippet: ST1926 induces S-phase arrest and apoptosis in colorectal cancer cells. A. ST1926 treatment causes accumulation of cells in the sub-G 1 region. B. Cell cycle distribution of colorectal cancer cells. HT29, HCT116, HCT116 p53 -/- , and HCT116 p21 -/- cells were treated with 0.1% DMSO or 1 μM ST1926 up to two days and stained with propidium iodide (50 mg/ml). The sub-G 1 percentage presumably indicates apoptotic cells. The sum of G 0 /G 1 , S, and G 2 /M phases is a percentage of nonapoptotic cells at days one and two post-ST1926 treatment. Percentage cells in the G 0 /G 1 phase are calculated as 100 - (S+G 2 /M). Results represent the average of three independent experiments (± SEM). C. TUNEL analysis of colorectal cancer cells treated with 0.1% DMSO or 1 μM ST1926 up to two days. Histograms are representative of two independent experiments and numbers indicate percentage of TUNEL-positive cells. D. ST1926 causes PARP cleavage in colorectal cancer cells. Cells were treated with 0.1% DMSO or 1 μM ST1926 up to two days. Total SDS protein lysates (50 µg/lane) were immunoblotted against PARP antibody. Arrow indicates cleaved PARP subunit. Similar trends were observed in three independent experiments. *P

    Article Snippet: Total cellular proteins (50 µg) were loaded onto a 10% SDS-polyacrylamide gel, subjected to electrophoresis and immunoblotted with the following antibodies: p53 (sc-126), p21 (sc-397), and PARP (sc-7150) from Santa Cruz Biotechnology (Heidelberg, Germany), γ-H2AX (2577) from Cell Signaling, Danvers, MA, POLA1 (31777) from Abcam (Cambridge, UK), and GAPDH (MAB5476) (Abnova, Heidelberg, Germany).

    Techniques: Staining, TUNEL Assay

    Immunoblotting with anti-Lyt51 antibodies. Lane 1, purified six-His-tagged Lyt51; lane 2, DN-001065; lane 3, mitomycin C-induced DN-001065; lane 4, CNRZ 302. The SDS cell extracts were prepared 40 min after mitomycin C induction of strain DN-001065. For each cell extract, the same protein amount was transferred on a nitrocellulose membrane. The blot was incubated with the purified antibodies directed against the six-His-tagged Lyt51 and then with protein G coupled to horseradish peroxidase. Positions of the six-His-tagged Lyt51 and the 31-kDa protein reacting with the anti-Lyt51 antibodies are indicated by arrowheads.

    Journal: Applied and Environmental Microbiology

    Article Title: The Streptococcus thermophilus Autolytic Phenotype Results from a Leaky Prophage

    doi:

    Figure Lengend Snippet: Immunoblotting with anti-Lyt51 antibodies. Lane 1, purified six-His-tagged Lyt51; lane 2, DN-001065; lane 3, mitomycin C-induced DN-001065; lane 4, CNRZ 302. The SDS cell extracts were prepared 40 min after mitomycin C induction of strain DN-001065. For each cell extract, the same protein amount was transferred on a nitrocellulose membrane. The blot was incubated with the purified antibodies directed against the six-His-tagged Lyt51 and then with protein G coupled to horseradish peroxidase. Positions of the six-His-tagged Lyt51 and the 31-kDa protein reacting with the anti-Lyt51 antibodies are indicated by arrowheads.

    Article Snippet: The same protein amount from each SDS cell extract and the purified His-tagged Lyt51 (2 ng) were electrophoresed on a 12.5% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell).

    Techniques: Purification, Incubation