streptavidin agarose beads  (Thermo Fisher)


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    Pierce High Capacity NeutrAvidin Agarose
    Description:
    Thermo Scientific High Capacity NeutrAvidin Agarose is a beaded agarose resin of immobilized NeutrAvidin Protein a modified form of avidin with exceptional biotin binding characteristics for affinity purification methods Features of High Capacity NeutrAvidin Agarose • NeutrAvidin protein purified deglycosylated avidin protein 60kDa pI 6 3 tetrameric with four biotin binding site per molecule • Agarose resin support is crosslinked 6 beaded agarose CL 6B the most popular resin for protein affinity purification methods • Inert and stable NeutrAvidin protein is immobilized by charge free leach resistant covalent bonds resulting in low nonspecific binding and enabling multiple uses without decline in yield • High capacity this variety of beads has a dense load of immobilized NeutrAvidin protein providing a binding capacity greater than 8 mg of biotinylated BSA per mL of resin High Capacity NeutrAvidin Agarose consists of a specially purified and deglycosylated form of avidin that has been covalently immobilized at a high density onto high quality crosslinked 6 beaded agarose Compared to native avidin NeutrAvidin protein is more neutral and exhibits much less nonspecific binding properties in biotin binding applications The agarose beads have physical and chemical properties that enable them to be used in a variety of batch or column type affinity procedures Generally NeutrAvidin products function as well or better than their streptavidin counterparts and are less expensive NeutrAvidin protein is deglycosylated native avidin from egg whites Removal of the excess carbohydrate by an exclusive process yields a protein with a more neutral isoelectric point and less nonspecific binding properties NeutrAvidin resins are prepared by covalently coupling the protein using efficient and stable chemistries resulting in supports that are resistant to leaching and stable at pH 2 11 The products are excellent choices for a variety of small or large scale affinity purification applications involving biotinylated macromolecules including separation of biotinylated molecules from samples and immunoprecipitation of antigens using biotin labeled antibodies Properties of crosslinked 6 beaded agarose CL 6B • Support pH Stability 2 to 14 short term 3 to 13 long term • Average Particle Size 45 to 165 microns • Exclusion Limit 10 000 to 4 000 000 daltons • Maximum Volumetric Flow Rate approx 1 mL minute for 1 cm diameter column • Maximum Linear Velocity 30 cm per hour • Maximum Pressure less than 25psi 1 5 bar Related Products Pierce NeutrAvidin Agarose Pierce NeutrAvidin UltraLink Resin Pierce NeutrAvidin Plus UltraLink Resin
    Catalog Number:
    29202
    Price:
    None
    Category:
    Chromatography Columns Resins Spin Filters
    Applications:
    Affinity Purification|Protein Biology|Protein Purification|Protein Purification & Isolation
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    Structured Review

    Thermo Fisher streptavidin agarose beads
    Insulin-induced translocation of GLUT4 is disrupted in muscle fibers isolated from HTZ mice. ( a–c ) Freshly dissected FDB muscles from 2 month-old WT and HTZ mice were digested with collagenase. Isolated fibers were stimulated during 15 min with 0.1 µM insulin to induce GLUT4 translocation, fixed and immunolabeled with a polyclonal-GLUT4 antibody. Translocation of GLUT4 was estimated by measuring the total intensity fluorescence of GLUT4 in ROIs at the sarcolemma. ( a ) Examples of the ROIs used are drawn in white. GLUT4 was measured on both edges of the confocal image and then averaged. ( b ) Examples images of GLUT4 signal in WT and HTZ fibers at the resting (left panels) and insulin-stimulated condition (right panels). Scale bar = 20 µm. ( c ) The graph show the averaged GLUT4 signal in sarcolemma. Note that insulin-induced GLUT4 translocation is significantly reduced in HTZ myofibers compared to WT myofibers. Data are expressed as mean GLUT4 fluorescence signal ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbols * and # denote significance with respect to WT-resting and WT-insulin-stimulated fibers, respectively. N is between 34 and 66 fibers from at least 10 different animals per genotype. ( d ) FDB muscles were dissected from WT and HTZ mice, stabilized in Tyrode solution, stimulated for 30 min with 0.1 µM insulin and then exposed to 1 mg/ml of biotin at 4 °C during 60 min. After quenching with 100 mM glycine, muscles were frozen and pulverized in liquid nitrogen, lysed and centrifuged at 14.000 g for 10 min. Supernatants were mixed with <t>streptavidin-agarose</t> beads overnight at 4 °C and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were used to evaluate GLUT4 expression by western blot. GAPDH was used as a control that only surface proteins were labeled in biotinylated fractions. On the left are shown representative blots per each condition, on the right are plotted the percentages of GLUT4 in biotinylated fractions. Data are expressed as mean GLUT4% ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbol * denote significance with respect to WT-muscles. N is five different animals per genotype.
    Thermo Scientific High Capacity NeutrAvidin Agarose is a beaded agarose resin of immobilized NeutrAvidin Protein a modified form of avidin with exceptional biotin binding characteristics for affinity purification methods Features of High Capacity NeutrAvidin Agarose • NeutrAvidin protein purified deglycosylated avidin protein 60kDa pI 6 3 tetrameric with four biotin binding site per molecule • Agarose resin support is crosslinked 6 beaded agarose CL 6B the most popular resin for protein affinity purification methods • Inert and stable NeutrAvidin protein is immobilized by charge free leach resistant covalent bonds resulting in low nonspecific binding and enabling multiple uses without decline in yield • High capacity this variety of beads has a dense load of immobilized NeutrAvidin protein providing a binding capacity greater than 8 mg of biotinylated BSA per mL of resin High Capacity NeutrAvidin Agarose consists of a specially purified and deglycosylated form of avidin that has been covalently immobilized at a high density onto high quality crosslinked 6 beaded agarose Compared to native avidin NeutrAvidin protein is more neutral and exhibits much less nonspecific binding properties in biotin binding applications The agarose beads have physical and chemical properties that enable them to be used in a variety of batch or column type affinity procedures Generally NeutrAvidin products function as well or better than their streptavidin counterparts and are less expensive NeutrAvidin protein is deglycosylated native avidin from egg whites Removal of the excess carbohydrate by an exclusive process yields a protein with a more neutral isoelectric point and less nonspecific binding properties NeutrAvidin resins are prepared by covalently coupling the protein using efficient and stable chemistries resulting in supports that are resistant to leaching and stable at pH 2 11 The products are excellent choices for a variety of small or large scale affinity purification applications involving biotinylated macromolecules including separation of biotinylated molecules from samples and immunoprecipitation of antigens using biotin labeled antibodies Properties of crosslinked 6 beaded agarose CL 6B • Support pH Stability 2 to 14 short term 3 to 13 long term • Average Particle Size 45 to 165 microns • Exclusion Limit 10 000 to 4 000 000 daltons • Maximum Volumetric Flow Rate approx 1 mL minute for 1 cm diameter column • Maximum Linear Velocity 30 cm per hour • Maximum Pressure less than 25psi 1 5 bar Related Products Pierce NeutrAvidin Agarose Pierce NeutrAvidin UltraLink Resin Pierce NeutrAvidin Plus UltraLink Resin
    https://www.bioz.com/result/streptavidin agarose beads/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
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    streptavidin agarose beads - by Bioz Stars, 2021-07
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    Images

    1) Product Images from "Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells"

    Article Title: Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-04418-w

    Insulin-induced translocation of GLUT4 is disrupted in muscle fibers isolated from HTZ mice. ( a–c ) Freshly dissected FDB muscles from 2 month-old WT and HTZ mice were digested with collagenase. Isolated fibers were stimulated during 15 min with 0.1 µM insulin to induce GLUT4 translocation, fixed and immunolabeled with a polyclonal-GLUT4 antibody. Translocation of GLUT4 was estimated by measuring the total intensity fluorescence of GLUT4 in ROIs at the sarcolemma. ( a ) Examples of the ROIs used are drawn in white. GLUT4 was measured on both edges of the confocal image and then averaged. ( b ) Examples images of GLUT4 signal in WT and HTZ fibers at the resting (left panels) and insulin-stimulated condition (right panels). Scale bar = 20 µm. ( c ) The graph show the averaged GLUT4 signal in sarcolemma. Note that insulin-induced GLUT4 translocation is significantly reduced in HTZ myofibers compared to WT myofibers. Data are expressed as mean GLUT4 fluorescence signal ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbols * and # denote significance with respect to WT-resting and WT-insulin-stimulated fibers, respectively. N is between 34 and 66 fibers from at least 10 different animals per genotype. ( d ) FDB muscles were dissected from WT and HTZ mice, stabilized in Tyrode solution, stimulated for 30 min with 0.1 µM insulin and then exposed to 1 mg/ml of biotin at 4 °C during 60 min. After quenching with 100 mM glycine, muscles were frozen and pulverized in liquid nitrogen, lysed and centrifuged at 14.000 g for 10 min. Supernatants were mixed with streptavidin-agarose beads overnight at 4 °C and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were used to evaluate GLUT4 expression by western blot. GAPDH was used as a control that only surface proteins were labeled in biotinylated fractions. On the left are shown representative blots per each condition, on the right are plotted the percentages of GLUT4 in biotinylated fractions. Data are expressed as mean GLUT4% ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbol * denote significance with respect to WT-muscles. N is five different animals per genotype.
    Figure Legend Snippet: Insulin-induced translocation of GLUT4 is disrupted in muscle fibers isolated from HTZ mice. ( a–c ) Freshly dissected FDB muscles from 2 month-old WT and HTZ mice were digested with collagenase. Isolated fibers were stimulated during 15 min with 0.1 µM insulin to induce GLUT4 translocation, fixed and immunolabeled with a polyclonal-GLUT4 antibody. Translocation of GLUT4 was estimated by measuring the total intensity fluorescence of GLUT4 in ROIs at the sarcolemma. ( a ) Examples of the ROIs used are drawn in white. GLUT4 was measured on both edges of the confocal image and then averaged. ( b ) Examples images of GLUT4 signal in WT and HTZ fibers at the resting (left panels) and insulin-stimulated condition (right panels). Scale bar = 20 µm. ( c ) The graph show the averaged GLUT4 signal in sarcolemma. Note that insulin-induced GLUT4 translocation is significantly reduced in HTZ myofibers compared to WT myofibers. Data are expressed as mean GLUT4 fluorescence signal ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbols * and # denote significance with respect to WT-resting and WT-insulin-stimulated fibers, respectively. N is between 34 and 66 fibers from at least 10 different animals per genotype. ( d ) FDB muscles were dissected from WT and HTZ mice, stabilized in Tyrode solution, stimulated for 30 min with 0.1 µM insulin and then exposed to 1 mg/ml of biotin at 4 °C during 60 min. After quenching with 100 mM glycine, muscles were frozen and pulverized in liquid nitrogen, lysed and centrifuged at 14.000 g for 10 min. Supernatants were mixed with streptavidin-agarose beads overnight at 4 °C and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were used to evaluate GLUT4 expression by western blot. GAPDH was used as a control that only surface proteins were labeled in biotinylated fractions. On the left are shown representative blots per each condition, on the right are plotted the percentages of GLUT4 in biotinylated fractions. Data are expressed as mean GLUT4% ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbol * denote significance with respect to WT-muscles. N is five different animals per genotype.

    Techniques Used: Translocation Assay, Isolation, Mouse Assay, Immunolabeling, Fluorescence, Expressing, Western Blot, Labeling

    2) Product Images from "Estrogen Regulation of Anti-Apoptotic Bcl-2 Family Member Mcl-1 Expression in Breast Cancer Cells"

    Article Title: Estrogen Regulation of Anti-Apoptotic Bcl-2 Family Member Mcl-1 Expression in Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0100364

    Estrogen increases ERα binding to specific region on Mcl-1 promoter. (A) Streptavidin pull-down assay to detect ER and transcription factor binding to a 50 bp double-stranded biotin labeled probe specific to Mcl-1 promoter region of interest (region 1). Cells were stimulated with estrogen (10 nM) and nuclear extracts were taken 6 and 24-hours post-estrogen treatment. Pull-down products were analyzed using SDS/polyacrylamide gel electrophoresis and western blotting. Both a scrambled probe and an excess of unlabeled probe were used as a control. Blot was probed with antibody specific for ERα. (B) Blot was probed with antibody specific for ERβ. (C) Blot was probed with antibody specific for Sp1. (D) Blot was probed with antibody specific for Sp3. (E) Relative accumulation of ERα protein expression, confirmed by densitometry. (F) Relative accumulation of Sp1 protein expression, confirmed by densitometry. (G) Schematic representation of Mcl-1 gene showing approximate locations of biotin labeled probes used for Streptavidin pull-down.
    Figure Legend Snippet: Estrogen increases ERα binding to specific region on Mcl-1 promoter. (A) Streptavidin pull-down assay to detect ER and transcription factor binding to a 50 bp double-stranded biotin labeled probe specific to Mcl-1 promoter region of interest (region 1). Cells were stimulated with estrogen (10 nM) and nuclear extracts were taken 6 and 24-hours post-estrogen treatment. Pull-down products were analyzed using SDS/polyacrylamide gel electrophoresis and western blotting. Both a scrambled probe and an excess of unlabeled probe were used as a control. Blot was probed with antibody specific for ERα. (B) Blot was probed with antibody specific for ERβ. (C) Blot was probed with antibody specific for Sp1. (D) Blot was probed with antibody specific for Sp3. (E) Relative accumulation of ERα protein expression, confirmed by densitometry. (F) Relative accumulation of Sp1 protein expression, confirmed by densitometry. (G) Schematic representation of Mcl-1 gene showing approximate locations of biotin labeled probes used for Streptavidin pull-down.

    Techniques Used: Binding Assay, Pull Down Assay, Labeling, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing

    Estrogen receptor fail to bind to other ERE half sites in the Mcl-1 promoter. Streptavidin pull-down assay to detect ER and transcription factor binding to a 50 bp double-stranded biotin labeled probe specific to Mcl-1 promoter region of interest (region 2). Cells were stimulated with estrogen (10 nM) and nuclear extracts were taken 6 and 24-hours post-estrogen treatment. Pull-down products were analyzed using SDS/polyacrylamide gel electrophoresis and western blotting. Both a scrambled probe and an excess of unlabeled probe were used as a control. Blot was probed with antibody specific for ERα. (B) Blot was probed with antibody specific for Sp1. (C) Streptavidin pull-down assay to detect ER and transcription factor binding to a 50 bp double-stranded biotin labeled probe specific to Mcl-1 promoter region of interest (region 3). Cells were stimulated with estrogen (10 nM) and nuclear extracts were taken 6 and 24-hours post-estrogen treatment. Both a scrambled probe and an excess of unlabeled probe were used as a control. Blot was probed with antibody specific for ERα. (D) Blot was probed with antibody specific for Sp1.
    Figure Legend Snippet: Estrogen receptor fail to bind to other ERE half sites in the Mcl-1 promoter. Streptavidin pull-down assay to detect ER and transcription factor binding to a 50 bp double-stranded biotin labeled probe specific to Mcl-1 promoter region of interest (region 2). Cells were stimulated with estrogen (10 nM) and nuclear extracts were taken 6 and 24-hours post-estrogen treatment. Pull-down products were analyzed using SDS/polyacrylamide gel electrophoresis and western blotting. Both a scrambled probe and an excess of unlabeled probe were used as a control. Blot was probed with antibody specific for ERα. (B) Blot was probed with antibody specific for Sp1. (C) Streptavidin pull-down assay to detect ER and transcription factor binding to a 50 bp double-stranded biotin labeled probe specific to Mcl-1 promoter region of interest (region 3). Cells were stimulated with estrogen (10 nM) and nuclear extracts were taken 6 and 24-hours post-estrogen treatment. Both a scrambled probe and an excess of unlabeled probe were used as a control. Blot was probed with antibody specific for ERα. (D) Blot was probed with antibody specific for Sp1.

    Techniques Used: Pull Down Assay, Binding Assay, Labeling, Polyacrylamide Gel Electrophoresis, Western Blot

    3) Product Images from "Withaferin A inhibits LFA-1-stimulated ZAP70 activity and T-cell motility"

    Article Title: Withaferin A inhibits LFA-1-stimulated ZAP70 activity and T-cell motility

    Journal: bioRxiv

    doi: 10.1101/2021.04.25.441369

    WFA binds to ZAP70 in human T-cells through covalent interactions. ( A ) Human primary T-cells pre-treated with WFA-Biotin for 3 h or DMSO were stimulated to migrate on rICAM-1-coated plates for 30 min and lysed. Cellular lysates were immunoprecipitated with streptavidin-coated agarose beads and then Western immunoblotted for ZAP70. Cellular lysates from DTT-treated cells were evaluated for WFA-Biotin binding as control, whereas T-cell lysates not mixed with WFA-Biotin were used as an in-put control. ( B ) LFA-1-stimulated T-cells either treated with DMSO or WFA-Biotin were immuno-stained with anti-ZAP70/Alexa Fluor® 488 ( green ), streptavidin/Alexa Fluor® 555 ( red ), phalloidin-Alexa Fluor® 647 (actin, cyan ) and Hoechst (nuclei, blue ) and then imaged by confocal microscopy. Data represent at least three independent experiments. Scale bar = 5 μM. ( C ) In silico analysis of WFA-ZAP70 interaction indicating hydrogen bonding and hydrophobic interactions.
    Figure Legend Snippet: WFA binds to ZAP70 in human T-cells through covalent interactions. ( A ) Human primary T-cells pre-treated with WFA-Biotin for 3 h or DMSO were stimulated to migrate on rICAM-1-coated plates for 30 min and lysed. Cellular lysates were immunoprecipitated with streptavidin-coated agarose beads and then Western immunoblotted for ZAP70. Cellular lysates from DTT-treated cells were evaluated for WFA-Biotin binding as control, whereas T-cell lysates not mixed with WFA-Biotin were used as an in-put control. ( B ) LFA-1-stimulated T-cells either treated with DMSO or WFA-Biotin were immuno-stained with anti-ZAP70/Alexa Fluor® 488 ( green ), streptavidin/Alexa Fluor® 555 ( red ), phalloidin-Alexa Fluor® 647 (actin, cyan ) and Hoechst (nuclei, blue ) and then imaged by confocal microscopy. Data represent at least three independent experiments. Scale bar = 5 μM. ( C ) In silico analysis of WFA-ZAP70 interaction indicating hydrogen bonding and hydrophobic interactions.

    Techniques Used: Immunoprecipitation, Western Blot, Binding Assay, Staining, Confocal Microscopy, In Silico

    4) Product Images from "Syntaxin 4 regulates the surface localization of a promyogenic receptor Cdo thereby promoting myogenic differentiation"

    Article Title: Syntaxin 4 regulates the surface localization of a promyogenic receptor Cdo thereby promoting myogenic differentiation

    Journal: Skeletal Muscle

    doi: 10.1186/s13395-015-0052-8

    The cell-surface-resident Cdo was specifically decreased in Stx4-depleted C2C12 cells. a C2C12 cells were transfected with pSuper or shStx4 expression vectors, and cells at the indicated differentiation time points were subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were also analyzed as control. b C2C12/pSuper or C2C12/shStx4 cells were transfected with Cdo-GFP expression vectors and subjected to immunostaining with N-Cadherin antibody, followed by confocal microscopy. The boxed area is shown as an enlarged view. The white arrows mark the area where the localization of Cdo-GFP under the N-Cadherin-resident cell surface is located. Size bar = 10 μm. c C2C12/pSuper or C2C12/shCdo cells were transfected with pcDNA or Stx4 expression vectors and subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were analyzed as control. d Control or shCdo-transfected C2C12 cells at D1 were subjected to surface biotinylation followed by streptavidin-bead pulldown and immunoblotting with indicated antibodies. e Control or shCdo expression vector transfected C2C12 cells were immunoprecipitated with antibody to GLUT4 and immunoblotted with antibodies to GLUT4, Stx4, and Cdo. f Stable C2C12 cells transfected with control, Cdo, or shCdo expression vector were incubated with or without 10 μg/ml insulin for 1 h, followed by 2-NBDG incubation for a further 1 h. Glucose uptake was measured by the relative fluorescence intensity. The experiment was repeated for three independent assays with similar results. Significant difference from insulin-incubated cells, * p
    Figure Legend Snippet: The cell-surface-resident Cdo was specifically decreased in Stx4-depleted C2C12 cells. a C2C12 cells were transfected with pSuper or shStx4 expression vectors, and cells at the indicated differentiation time points were subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were also analyzed as control. b C2C12/pSuper or C2C12/shStx4 cells were transfected with Cdo-GFP expression vectors and subjected to immunostaining with N-Cadherin antibody, followed by confocal microscopy. The boxed area is shown as an enlarged view. The white arrows mark the area where the localization of Cdo-GFP under the N-Cadherin-resident cell surface is located. Size bar = 10 μm. c C2C12/pSuper or C2C12/shCdo cells were transfected with pcDNA or Stx4 expression vectors and subjected to the surface biotin labeling, followed by the pulldown with streptavidin and immunoblotting. Total cell lysates were analyzed as control. d Control or shCdo-transfected C2C12 cells at D1 were subjected to surface biotinylation followed by streptavidin-bead pulldown and immunoblotting with indicated antibodies. e Control or shCdo expression vector transfected C2C12 cells were immunoprecipitated with antibody to GLUT4 and immunoblotted with antibodies to GLUT4, Stx4, and Cdo. f Stable C2C12 cells transfected with control, Cdo, or shCdo expression vector were incubated with or without 10 μg/ml insulin for 1 h, followed by 2-NBDG incubation for a further 1 h. Glucose uptake was measured by the relative fluorescence intensity. The experiment was repeated for three independent assays with similar results. Significant difference from insulin-incubated cells, * p

    Techniques Used: Transfection, Expressing, Labeling, Immunostaining, Confocal Microscopy, Plasmid Preparation, Immunoprecipitation, Incubation, Fluorescence

    5) Product Images from "Glucocorticoids reduce renal NHE8 expression"

    Article Title: Glucocorticoids reduce renal NHE8 expression

    Journal: Physiological Reports

    doi: 10.1002/phy2.31

    Effect of dexamethasone on NHE8 surface protein abundance in NRK cells. Confluent NRK cells were processed for total protein abundance (A) or were surface biotinylated and surface proteins harvested using streptavidin-agarose beads (B). NHE8 total protein and surface protein abundance was less in dexamethasone-treated cells than control.
    Figure Legend Snippet: Effect of dexamethasone on NHE8 surface protein abundance in NRK cells. Confluent NRK cells were processed for total protein abundance (A) or were surface biotinylated and surface proteins harvested using streptavidin-agarose beads (B). NHE8 total protein and surface protein abundance was less in dexamethasone-treated cells than control.

    Techniques Used:

    6) Product Images from "Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking"

    Article Title: Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200709177

    TM5b overexpression inhibits AQP2 trafficking in MDCK/AQP2 cells. (A) MDCK/AQP2 cells were transfected with GFP-TM5b or GFP (green) and treated without (top) or with (bottom) forskolin. Cells were labeled for AQP2 (red). Bars, 10 μm. (B and C) Apical cell surface biotinylation assay using MDCK/AQP2 cells transfected with GFP-TM5b or GFP, corresponding to A. (B) Biotinylated proteins were precipitated with streptavidin-agarose beads and immunoblotted for AQP2. (C) The densitometric quantification normalized to GFP-transfected cells without forskolin stimulation. Data represent the mean and SE from three independent experiments. *, P
    Figure Legend Snippet: TM5b overexpression inhibits AQP2 trafficking in MDCK/AQP2 cells. (A) MDCK/AQP2 cells were transfected with GFP-TM5b or GFP (green) and treated without (top) or with (bottom) forskolin. Cells were labeled for AQP2 (red). Bars, 10 μm. (B and C) Apical cell surface biotinylation assay using MDCK/AQP2 cells transfected with GFP-TM5b or GFP, corresponding to A. (B) Biotinylated proteins were precipitated with streptavidin-agarose beads and immunoblotted for AQP2. (C) The densitometric quantification normalized to GFP-transfected cells without forskolin stimulation. Data represent the mean and SE from three independent experiments. *, P

    Techniques Used: Over Expression, Transfection, Labeling, Cell Surface Biotinylation Assay

    Apical F-actin is regulated by TM5b. (A) MDCK cells transfected with TM5b siRNA or scrambled control siRNA, which was indicated by fluorescein-labeled dsRNA (red), were treated without (top) or with (bottom) forskolin and labeled for F-actin (green). (B) MDCK cells transfected with GFP-TM5b or GFP (green) were treated without (top) or with (bottom) forskolin and labeled for F-actin (red). (C and D) Quantification of F-actin associated with the apical membrane. (C) MDCK cells transfected with TM5b siRNA or control siRNA were treated with or without forskolin and biotinylated on the apical surface. The lysates were incubated with phalloidin conjugated with Alexa Fluor 488 and precipitated with streptavidin-agarose beads and the fluorescence was measured. Each value was normalized to control siRNA-transfected cells without forskolin. Data represent the mean and SE from three independent experiments. *, P
    Figure Legend Snippet: Apical F-actin is regulated by TM5b. (A) MDCK cells transfected with TM5b siRNA or scrambled control siRNA, which was indicated by fluorescein-labeled dsRNA (red), were treated without (top) or with (bottom) forskolin and labeled for F-actin (green). (B) MDCK cells transfected with GFP-TM5b or GFP (green) were treated without (top) or with (bottom) forskolin and labeled for F-actin (red). (C and D) Quantification of F-actin associated with the apical membrane. (C) MDCK cells transfected with TM5b siRNA or control siRNA were treated with or without forskolin and biotinylated on the apical surface. The lysates were incubated with phalloidin conjugated with Alexa Fluor 488 and precipitated with streptavidin-agarose beads and the fluorescence was measured. Each value was normalized to control siRNA-transfected cells without forskolin. Data represent the mean and SE from three independent experiments. *, P

    Techniques Used: Transfection, Labeling, Incubation, Fluorescence

    TM5b knockdown promotes AQP2 trafficking in MDCK/AQP2 cells. (A–D) MDCK/AQP2 cells were transfected with TM5b siRNA (B) or scrambled control siRNA (A), which was indicated by fluorescein-labeled dsRNA (red), and treated without (top) or with (bottom) forskolin. Pretreatment with H89 was also performed using the cells transfected with TM5b siRNA (D) or the control siRNA (C). Cells were labeled for AQP2 (green). Bars, 10 μm. (E and F) Apical cell surface biotinylation assay using MDCK/AQP2 cells transfected with TM5b siRNA or control siRNA, corresponding to A–D. (E) Biotinylated proteins were precipitated with streptavidin-agarose beads and immunoblotted for AQP2. (F) The densitometric quantification normalized to control siRNA-transfected cells without forskolin treatment. Data represent the mean and SE from three independent experiments. *, P
    Figure Legend Snippet: TM5b knockdown promotes AQP2 trafficking in MDCK/AQP2 cells. (A–D) MDCK/AQP2 cells were transfected with TM5b siRNA (B) or scrambled control siRNA (A), which was indicated by fluorescein-labeled dsRNA (red), and treated without (top) or with (bottom) forskolin. Pretreatment with H89 was also performed using the cells transfected with TM5b siRNA (D) or the control siRNA (C). Cells were labeled for AQP2 (green). Bars, 10 μm. (E and F) Apical cell surface biotinylation assay using MDCK/AQP2 cells transfected with TM5b siRNA or control siRNA, corresponding to A–D. (E) Biotinylated proteins were precipitated with streptavidin-agarose beads and immunoblotted for AQP2. (F) The densitometric quantification normalized to control siRNA-transfected cells without forskolin treatment. Data represent the mean and SE from three independent experiments. *, P

    Techniques Used: Transfection, Labeling, Cell Surface Biotinylation Assay

    RNAi-mediated knockdown of TM5b promotes AQP2 trafficking, whereas overexpression of TM5b inhibits the AQP2 trafficking. (A and B) MDCK cells were transfected with cDNA encoding GFP-AQP2 (green) and with either TM5b siRNA (B) or scrambled control siRNA (A), which was indicated by fluorescein-labeled double-stranded RNA (dsRNA; red), and stimulated with forskolin. (C) MDCK cells were transfected with DsRed-AQP2 (red) and GFP-TM5b (green) and stimulated with forskolin. The bottoms represent z scans at the positions indicated by the white lines in the xy planes. Bars, 10 μm. (D and E) Apical cell surface biotinylation assay using the cells transfected with GFP-AQP2 and with TM5b siRNA or scrambled control siRNA, corresponding to A and B. No siRNA indicates the cells transfected with GFP-AQP2 alone. (D) Biotinylated proteins were precipitated with streptavidin-agarose beads and immunoblotted for AQP2. (E) The densitometric quantification normalized to the cells without siRNA and forskolin treatment. Data represent the mean and SE from three independent experiments. *, P
    Figure Legend Snippet: RNAi-mediated knockdown of TM5b promotes AQP2 trafficking, whereas overexpression of TM5b inhibits the AQP2 trafficking. (A and B) MDCK cells were transfected with cDNA encoding GFP-AQP2 (green) and with either TM5b siRNA (B) or scrambled control siRNA (A), which was indicated by fluorescein-labeled double-stranded RNA (dsRNA; red), and stimulated with forskolin. (C) MDCK cells were transfected with DsRed-AQP2 (red) and GFP-TM5b (green) and stimulated with forskolin. The bottoms represent z scans at the positions indicated by the white lines in the xy planes. Bars, 10 μm. (D and E) Apical cell surface biotinylation assay using the cells transfected with GFP-AQP2 and with TM5b siRNA or scrambled control siRNA, corresponding to A and B. No siRNA indicates the cells transfected with GFP-AQP2 alone. (D) Biotinylated proteins were precipitated with streptavidin-agarose beads and immunoblotted for AQP2. (E) The densitometric quantification normalized to the cells without siRNA and forskolin treatment. Data represent the mean and SE from three independent experiments. *, P

    Techniques Used: Over Expression, Transfection, Labeling, Cell Surface Biotinylation Assay

    7) Product Images from "Juvenile Cataract-Associated Mutation of Solute Carrier SLC16A12 Impairs Trafficking of the Protein to the Plasma Membrane"

    Article Title: Juvenile Cataract-Associated Mutation of Solute Carrier SLC16A12 Impairs Trafficking of the Protein to the Plasma Membrane

    Journal: Investigative Ophthalmology & Visual Science

    doi: 10.1167/iovs.10-6579

    MCT12:214Δ is retained in the ER. ( A ) HEK-293 cells transiently transfected with vectors expressing either MCT12:214Δ-GFP ( upper ) or MCT12-GFP ( lower ) were immunostained for GRP78 ( red ). Scale bars, 10 μm. ( B ) HEK-293 cells transiently transfected with vectors expressing either MCT12-GFP or MCT12:214Δ-GFP were cell-surface biotinylated 48 hours after transfection. Biotinylated proteins were precipitated from cell lysates with streptavidin-agarose beads. Soluble (S) and avidin-precipitated (Av) fractions were run by SDS-PAGE and subjected to Western blot analysis, and the blots were probed for GFP and CD147. ( C ) To determine whether differences in protein levels between MCT12-GFP and MCT12:214Δ-GFP were due to differences in mRNA levels, mRNA was extracted from HEK-293 cells transiently transfected with either MCT12-GFP or MCT12:214Δ-GFP 48 hours after transfection, and cDNA was prepared. PCR was performed for GFP to unify amplification from both constructs and β-actin as a control.
    Figure Legend Snippet: MCT12:214Δ is retained in the ER. ( A ) HEK-293 cells transiently transfected with vectors expressing either MCT12:214Δ-GFP ( upper ) or MCT12-GFP ( lower ) were immunostained for GRP78 ( red ). Scale bars, 10 μm. ( B ) HEK-293 cells transiently transfected with vectors expressing either MCT12-GFP or MCT12:214Δ-GFP were cell-surface biotinylated 48 hours after transfection. Biotinylated proteins were precipitated from cell lysates with streptavidin-agarose beads. Soluble (S) and avidin-precipitated (Av) fractions were run by SDS-PAGE and subjected to Western blot analysis, and the blots were probed for GFP and CD147. ( C ) To determine whether differences in protein levels between MCT12-GFP and MCT12:214Δ-GFP were due to differences in mRNA levels, mRNA was extracted from HEK-293 cells transiently transfected with either MCT12-GFP or MCT12:214Δ-GFP 48 hours after transfection, and cDNA was prepared. PCR was performed for GFP to unify amplification from both constructs and β-actin as a control.

    Techniques Used: Transfection, Expressing, Avidin-Biotin Assay, SDS Page, Western Blot, Polymerase Chain Reaction, Amplification, Construct

    8) Product Images from "Arsenite Targets the RING Finger Domain of Rbx1 E3 Ubiquitin Ligase to Inhibit Proteasome-Mediated Degradation of Nrf2"

    Article Title: Arsenite Targets the RING Finger Domain of Rbx1 E3 Ubiquitin Ligase to Inhibit Proteasome-Mediated Degradation of Nrf2

    Journal: Chemical research in toxicology

    doi: 10.1021/acs.chemrestox.8b00062

    Arsenite binds to the Rbx1 protein in cells. (a) The chemical structure of the biotin-As probe. (b) Streptavidin agarose affinity pull-down assay indicating the interaction between As 3+ and Rbx1 in cells. The biotin-As probe was used to pull down ectopically expressed HA-Rbx1 in HEK293T cells. The HA-Rbx1 signal was detected using the anti-HA antibody, and the input HA-Rbx1 and actin were also detected. (c,d) The interaction between the biotin-As probe and Rbx1 was substantially diminished upon pretreatment with 10 μM NaAsO 2 , PAPAO, and to a lesser extent, 10 μM Zn 2+ . The Western blot images are shown in (c), and the quantification results are displayed in (d). The data represent the means and standard deviations of results obtained from 3 biological replicates. The P values (for comparisons with the control, unless otherwise indicated) were calculated using an unpaired two-tailed student’s t-test (*, 0.01
    Figure Legend Snippet: Arsenite binds to the Rbx1 protein in cells. (a) The chemical structure of the biotin-As probe. (b) Streptavidin agarose affinity pull-down assay indicating the interaction between As 3+ and Rbx1 in cells. The biotin-As probe was used to pull down ectopically expressed HA-Rbx1 in HEK293T cells. The HA-Rbx1 signal was detected using the anti-HA antibody, and the input HA-Rbx1 and actin were also detected. (c,d) The interaction between the biotin-As probe and Rbx1 was substantially diminished upon pretreatment with 10 μM NaAsO 2 , PAPAO, and to a lesser extent, 10 μM Zn 2+ . The Western blot images are shown in (c), and the quantification results are displayed in (d). The data represent the means and standard deviations of results obtained from 3 biological replicates. The P values (for comparisons with the control, unless otherwise indicated) were calculated using an unpaired two-tailed student’s t-test (*, 0.01

    Techniques Used: Pull Down Assay, Western Blot, Two Tailed Test

    9) Product Images from "Targeted inactivation of MLL3 histone H3-Lys-4 methyltransferase activity in the mouse reveals vital roles for MLL3 in adipogenesis"

    Article Title: Targeted inactivation of MLL3 histone H3-Lys-4 methyltransferase activity in the mouse reveals vital roles for MLL3 in adipogenesis

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0810100105

    Direct interaction of ASCOM with promoter-free and promoter-bound PPARγ·RXRα. ( A ) Purified ASCOM complex. The immuno-affinity purified complex was analyzed by SDS/PAGE (4–20% gradient) with silver staining. Polypeptides identified by mass spectrometry are indicated. ( B ) ASCOM binding to promoter-free PPARγ·RXRα. The purified complex was incubated with M2 agarose (lane 2) or M2 agarose-bound FLAG-PPARγ·RXRα in the absence (lane 3) or presence (lane 4) of 5.0 μM 15d-deoxy-Δ 12,14 -Prostagladin J 2 . ( C ) ASCOM binding to promoter-bound PPARγ·RXRα. The purified complex was incubated with a Streptavidin agarose-immobilized biotinylated DNA fragment (containing 3 DR1 sites) in the absence (lane 2) or presence (lanes 3–5) of PPARγ and RXRα and either with no ligand (lanes 2 and 3) or with 5.0 μM 15d-deoxy-Δ 12,14 -prostagladin J 2 (lane 4) or 5.0 μM rosiglitazone (lane 5). Bound proteins in B and C were detected by immunoblot (ASC-2, ASH2, RbBP5, and WDR5) or staining (PPARγ and RXRα). Lanes 1 in B and C contained 10% of the input samples.
    Figure Legend Snippet: Direct interaction of ASCOM with promoter-free and promoter-bound PPARγ·RXRα. ( A ) Purified ASCOM complex. The immuno-affinity purified complex was analyzed by SDS/PAGE (4–20% gradient) with silver staining. Polypeptides identified by mass spectrometry are indicated. ( B ) ASCOM binding to promoter-free PPARγ·RXRα. The purified complex was incubated with M2 agarose (lane 2) or M2 agarose-bound FLAG-PPARγ·RXRα in the absence (lane 3) or presence (lane 4) of 5.0 μM 15d-deoxy-Δ 12,14 -Prostagladin J 2 . ( C ) ASCOM binding to promoter-bound PPARγ·RXRα. The purified complex was incubated with a Streptavidin agarose-immobilized biotinylated DNA fragment (containing 3 DR1 sites) in the absence (lane 2) or presence (lanes 3–5) of PPARγ and RXRα and either with no ligand (lanes 2 and 3) or with 5.0 μM 15d-deoxy-Δ 12,14 -prostagladin J 2 (lane 4) or 5.0 μM rosiglitazone (lane 5). Bound proteins in B and C were detected by immunoblot (ASC-2, ASH2, RbBP5, and WDR5) or staining (PPARγ and RXRα). Lanes 1 in B and C contained 10% of the input samples.

    Techniques Used: Purification, Affinity Purification, SDS Page, Silver Staining, Mass Spectrometry, Binding Assay, Incubation, Staining

    10) Product Images from "Functional interaction between p75NTR and TrkA: the endocytic trafficking of p75NTR is driven by TrkA and regulates TrkA-mediated signalling"

    Article Title: Functional interaction between p75NTR and TrkA: the endocytic trafficking of p75NTR is driven by TrkA and regulates TrkA-mediated signalling

    Journal: Biochemical Journal

    doi: 10.1042/BJ20041155

    Stimulation of TrkA by the basolateral addition of NGF induces p75 NTR transcytosis at the basolateral surface ( A ) E1A5 cells were stimulated by adding NGF from the basolateral surface. Cells were biotinylated with NHS-SS-biotin as described in the Materials and methods section. Protein extracts from treated and control cells were immunoprecipitated with streptavidin. Immunocomplexes (BIOT) and aliquots comprising 1/10 of the total extracts were separated on SDS/8%-PAGE. Western blot analysis was carried out using an anti-TrkA antibody. Glut., glutathione. ( B ) E1A5 cells were stimulated with NGF added from the basolateral surface for the times indicated. Treated and control filters were subjected to indirect immunofluorescence using an anti-p75 NTR monoclonal antibody and analysed by confocal microscopy. ( C ) E1A5 cells were stimulated as described in ( B ). Protein extracts from treated and control cells were immunoprecipitated with anti-p75 NTR antibody. Western blot analysis was carried out using an anti-TrkA (upper) or an anti-p75 NTR (lower) antibody. ( D ) E1A5 cells were stimulated for 15 min by basolateral addition of NGF. The filters were divided into two parts and subjected to indirect immunofluorescence with anti-p75 NTR antibody in either the absence or the presence of the detergent saponin, in order to detect only cell surface (in the absence of saponin) or both cell surface and intracellular compartments. These experiments were repeated more than three times, with similar results.
    Figure Legend Snippet: Stimulation of TrkA by the basolateral addition of NGF induces p75 NTR transcytosis at the basolateral surface ( A ) E1A5 cells were stimulated by adding NGF from the basolateral surface. Cells were biotinylated with NHS-SS-biotin as described in the Materials and methods section. Protein extracts from treated and control cells were immunoprecipitated with streptavidin. Immunocomplexes (BIOT) and aliquots comprising 1/10 of the total extracts were separated on SDS/8%-PAGE. Western blot analysis was carried out using an anti-TrkA antibody. Glut., glutathione. ( B ) E1A5 cells were stimulated with NGF added from the basolateral surface for the times indicated. Treated and control filters were subjected to indirect immunofluorescence using an anti-p75 NTR monoclonal antibody and analysed by confocal microscopy. ( C ) E1A5 cells were stimulated as described in ( B ). Protein extracts from treated and control cells were immunoprecipitated with anti-p75 NTR antibody. Western blot analysis was carried out using an anti-TrkA (upper) or an anti-p75 NTR (lower) antibody. ( D ) E1A5 cells were stimulated for 15 min by basolateral addition of NGF. The filters were divided into two parts and subjected to indirect immunofluorescence with anti-p75 NTR antibody in either the absence or the presence of the detergent saponin, in order to detect only cell surface (in the absence of saponin) or both cell surface and intracellular compartments. These experiments were repeated more than three times, with similar results.

    Techniques Used: Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Immunofluorescence, Confocal Microscopy

    Stimulation of p75 NTR by the apical addition of NGF induces p75 NTR transcytosis at the basolateral surface ( A ) Upper panel: immunoblotting with anti-TrkA antibodies against the N-terminus (left) and against a C-terminal peptide (right). Lower panel: FRT cells expressing p75 NTR (E1A5 clone) were grown on filters for 5 days and then subjected to indirect immunofluorescence using anti-TrkA or anti-p75 NTR antibodies as indicated in the Materials and methods section. A, apical membrane; BL, basolateral membrane. ( B ) E1A5 cells were biotinylated with NHS-SS-biotin (BIOT.) as described in the Material and methods section. Immunocomplexes and 1/10 of the total extracts were subjected to Western blot analysis using an anti-p75 NTR antibody. Glut., glutathione. ( C ) Treated and control filters were divided into four parts and subjected to indirect immunofluorescence carried out using the appropriate antibodies as indicated. ( D ) Proteins were labelled in vivo using [ 35 S]Met and [ 35 S]Cys for 14 h. After p75 NTR -specific stimulation with NGF, surface proteins were labelled from the apical or basolateral surfaces with biotin as described in the Materials and methods section. Proteins were immunoprecipitated with the appropriate antibody, boiled in 0.5% SDS, diluted in lysis solution, and immunoprecipitated again with streptavidin to identify the biotinylated fraction. Proteins were separated by SDS/PAGE, dried and exposed to autoradiography. Aliquots containing 1/10 of the total immunoprecipitated proteins are shown as a control on the right-hand side. These experiments are representative of at least three independent experiments.
    Figure Legend Snippet: Stimulation of p75 NTR by the apical addition of NGF induces p75 NTR transcytosis at the basolateral surface ( A ) Upper panel: immunoblotting with anti-TrkA antibodies against the N-terminus (left) and against a C-terminal peptide (right). Lower panel: FRT cells expressing p75 NTR (E1A5 clone) were grown on filters for 5 days and then subjected to indirect immunofluorescence using anti-TrkA or anti-p75 NTR antibodies as indicated in the Materials and methods section. A, apical membrane; BL, basolateral membrane. ( B ) E1A5 cells were biotinylated with NHS-SS-biotin (BIOT.) as described in the Material and methods section. Immunocomplexes and 1/10 of the total extracts were subjected to Western blot analysis using an anti-p75 NTR antibody. Glut., glutathione. ( C ) Treated and control filters were divided into four parts and subjected to indirect immunofluorescence carried out using the appropriate antibodies as indicated. ( D ) Proteins were labelled in vivo using [ 35 S]Met and [ 35 S]Cys for 14 h. After p75 NTR -specific stimulation with NGF, surface proteins were labelled from the apical or basolateral surfaces with biotin as described in the Materials and methods section. Proteins were immunoprecipitated with the appropriate antibody, boiled in 0.5% SDS, diluted in lysis solution, and immunoprecipitated again with streptavidin to identify the biotinylated fraction. Proteins were separated by SDS/PAGE, dried and exposed to autoradiography. Aliquots containing 1/10 of the total immunoprecipitated proteins are shown as a control on the right-hand side. These experiments are representative of at least three independent experiments.

    Techniques Used: Expressing, Immunofluorescence, Western Blot, In Vivo, Immunoprecipitation, Lysis, SDS Page, Autoradiography

    11) Product Images from "STING Recognition of Cytoplasmic DNA Instigates Cellular Defense"

    Article Title: STING Recognition of Cytoplasmic DNA Instigates Cellular Defense

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2013.01.039

    (A) hTERT-BJ1 cells were transfected with biotin conjugated dsDNA90 (3 μg/ml) for 6h and treated with DSS. Lysates were precipitated using streptavidin agarose beads and analyzed by immunoblotting using anti-HA antibody. (B) hTERT-BJ1 cells were transfected with biotin conjugated dsDNA90 (3 μg/ml) for 6h and treated with UV. Same as A . (C) Sting +/+ or Sting -/- MEFs were transfected with biotin conjugated dsDNA45 and crosslinked by UV. Lysates were precipitated by streptavidin agarose beads and analyzed by immunoblotting.
    Figure Legend Snippet: (A) hTERT-BJ1 cells were transfected with biotin conjugated dsDNA90 (3 μg/ml) for 6h and treated with DSS. Lysates were precipitated using streptavidin agarose beads and analyzed by immunoblotting using anti-HA antibody. (B) hTERT-BJ1 cells were transfected with biotin conjugated dsDNA90 (3 μg/ml) for 6h and treated with UV. Same as A . (C) Sting +/+ or Sting -/- MEFs were transfected with biotin conjugated dsDNA45 and crosslinked by UV. Lysates were precipitated by streptavidin agarose beads and analyzed by immunoblotting.

    Techniques Used: Transfection

    12) Product Images from "PKCα regulates vasopressin-induced aquaporin-2 trafficking in mouse kidney collecting duct cells in vitro via altering microtubule assembly"

    Article Title: PKCα regulates vasopressin-induced aquaporin-2 trafficking in mouse kidney collecting duct cells in vitro via altering microtubule assembly

    Journal: Acta Pharmacologica Sinica

    doi: 10.1038/aps.2011.160

    Regulation of PKCα expression influenced DdAVP stimulated plasma location of AQP2. (A) AQP2-GFP stably expressed WT7 cells were transfected with indicated constructs. Forty-eight hours after transfection, surface membrane proteins were biotinylated at 0 min, DdAVP treated 30 min, and DdAVP treated 30 min followed by 2 h washout. Whole cell lysates were subjected to avidin pulldown assay using streptavidin-agarose beads or immunoprecipitation (IP) using anti-AQP2 antibodies. The recovered proteins were separated by SDS-PAGE and analyzed by Western blot for AQP2 as described in Materials and Methods. For each condition, the detected signals from avidin-pulldown assay and AQP2 immunoprecipitation were from same SDS-PAGE gel. (B) Signals from 3 independent experiments were quantified by densitometry. The intensity of the AQP2 signals from the avidin pulldowns were normalized to the corresponding AQP2 IP signals under each condition. Data were represented as mean±SEM. b P
    Figure Legend Snippet: Regulation of PKCα expression influenced DdAVP stimulated plasma location of AQP2. (A) AQP2-GFP stably expressed WT7 cells were transfected with indicated constructs. Forty-eight hours after transfection, surface membrane proteins were biotinylated at 0 min, DdAVP treated 30 min, and DdAVP treated 30 min followed by 2 h washout. Whole cell lysates were subjected to avidin pulldown assay using streptavidin-agarose beads or immunoprecipitation (IP) using anti-AQP2 antibodies. The recovered proteins were separated by SDS-PAGE and analyzed by Western blot for AQP2 as described in Materials and Methods. For each condition, the detected signals from avidin-pulldown assay and AQP2 immunoprecipitation were from same SDS-PAGE gel. (B) Signals from 3 independent experiments were quantified by densitometry. The intensity of the AQP2 signals from the avidin pulldowns were normalized to the corresponding AQP2 IP signals under each condition. Data were represented as mean±SEM. b P

    Techniques Used: Expressing, Stable Transfection, Transfection, Construct, Avidin-Biotin Assay, Immunoprecipitation, SDS Page, Western Blot

    13) Product Images from "FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane"

    Article Title: FKBP8 Enhances Protein Stability of the CLC-1 Chloride Channel at the Plasma Membrane

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19123783

    Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing Flag-CLC-1 in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p
    Figure Legend Snippet: Detection of cell surface FKBP8 expression by biotinylation. ( A ) Surface biotinylation experiments on HEK293T cells expressing Flag-CLC-1 in the absence or presence of Myc-FKBP8. ( Left ) Representative immunoblots. Co-expression with the Myc vector was used as the vector control (-). Cell lysates from biotinylated intact cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ), using the indicated antibodies (α-Myc, α-Flag, or α-GAPDH). Total represents about 8% of the amount of the protein used for streptavidin pull-down. The molecular weight markers (in kDa) are labeled to the left. GAPDH expressions are shown as the loading control. ( Right ) Quantification of total and surface CLC-1 protein levels ( n = 6). The protein density was normalized to that of the corresponding vector control; ( B ) Surface biotinylation analysis of Myc-FKBP8 in the absence or presence of Flag-CLC-1. ( Left ) Representative immunoblots. ( Right ) Quantification of FKBP8 protein levels and membrane trafficking ( n = 8). The membrane trafficking efficiency of FKBP8 was expressed as surface protein density divided by the corresponding total protein density ( Surface/total ); ( C ) Representative immunoblots showing surface biotinylation analysis of Myc-Aha1, HA-HOP, HA-Hsp90β, or Myc-Hsc70, in the absence or presence of Flag-CLC-1. Asterisks denote significant difference from the control (*, t -test: p

    Techniques Used: Expressing, Western Blot, Plasmid Preparation, Molecular Weight, Labeling

    14) Product Images from "Phospholipid flippase ATP11C is endocytosed and downregulated following Ca2+-mediated protein kinase C activation"

    Article Title: Phospholipid flippase ATP11C is endocytosed and downregulated following Ca2+-mediated protein kinase C activation

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01338-1

    Flippase activity of ATP11C inhibited by PKC is partially rescued by endocytosis-defective mutants. a – d Ba/F3 cells stably expressing the indicated point mutants of ATP11C-HA were treated with vehicle alone (Mock), 400 nM PMA (PMA), or 1 μM A23187 in the presence of CaCl 2 (A23187) for 15 min. a Cells were immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 20 μm. b The cells with ATP11C mutants localized to the plasma membrane (PM), to the plasma membrane and endosomes (PM + E), or to the endosomes (E) were counted; counts were normalized against the total number of counted cells. In each sample, 315–459 cells were counted. Graphs display averages ± SD from three independent experiments. c Cell-surface expression level of ATP11A, ATP11C, and the indicated mutants upon treatment with PMA were analyzed after surface biotinylation. Proteins precipitated with streptavidin-agarose beads were subjected to immunoblot analysis (upper panels, biotinylated). Ten percent of the input of the biotinylation reaction was loaded in each lane (lower panels, total lysate). Expression levels of P4-ATPases were analyzed by immunoblotting with anti-HA and anti-ATP1A1 (as an internal control) antibodies. d Relative surface expression levels of proteins, normalized against the level of ATP1A1 (used as an internal control). Graphs display averages ± SD from four independent experiments. ** p
    Figure Legend Snippet: Flippase activity of ATP11C inhibited by PKC is partially rescued by endocytosis-defective mutants. a – d Ba/F3 cells stably expressing the indicated point mutants of ATP11C-HA were treated with vehicle alone (Mock), 400 nM PMA (PMA), or 1 μM A23187 in the presence of CaCl 2 (A23187) for 15 min. a Cells were immunostained with anti-HA and anti-ATP1A1 antibodies, followed by incubation with Cy3-conjugated anti-rat and Alexa Fluor 488-conjugated anti-rabbit secondary antibodies. Scale bars, 20 μm. b The cells with ATP11C mutants localized to the plasma membrane (PM), to the plasma membrane and endosomes (PM + E), or to the endosomes (E) were counted; counts were normalized against the total number of counted cells. In each sample, 315–459 cells were counted. Graphs display averages ± SD from three independent experiments. c Cell-surface expression level of ATP11A, ATP11C, and the indicated mutants upon treatment with PMA were analyzed after surface biotinylation. Proteins precipitated with streptavidin-agarose beads were subjected to immunoblot analysis (upper panels, biotinylated). Ten percent of the input of the biotinylation reaction was loaded in each lane (lower panels, total lysate). Expression levels of P4-ATPases were analyzed by immunoblotting with anti-HA and anti-ATP1A1 (as an internal control) antibodies. d Relative surface expression levels of proteins, normalized against the level of ATP1A1 (used as an internal control). Graphs display averages ± SD from four independent experiments. ** p

    Techniques Used: Activity Assay, Stable Transfection, Expressing, Incubation

    ATP11C is endocytosed by treatment with phorbol 12-myristate 13-acetate (PMA) and increasing cytosolic Ca 2+ . a HeLa cells stably expressing C-terminally HA-tagged ATP11A, and ATP11C were treated for 15 min at 37 °C with vehicle alone (Mock); with either 400 nM of PMA (PMA) or PMA and 2 μM of BIM-1 (PMA + BIM); or with 1 μM A23187 in the presence of either 1.8 mM of CaCl 2 (A23187) or 1.8 mM CaCl 2 and 2 μM BIM-1 (A23187 + BIM). The cells were fixed and immunostained with anti-HA antibody, followed by Cy3-conjugated anti-rat secondary antibody. See Supplementary Fig. 1 and Supplementary Movie 1 . b Cell-surface expression levels of ATP11A and ATP11C following treatment with PMA or A23187 and CaCl 2 were analyzed after surface biotinylation. Proteins precipitated with streptavidin-agarose beads were subjected to immunoblot analysis (left panels, biotinylated). 15% of the input of the biotinylation reaction was loaded in each lane (right panels, total lysate). Expression of ATP11A and ATP11C proteins was analyzed by immunoblotting with anti-HA and anti-ATP1A1 (as an internal control) antibodies. c HeLa cells stably expressing HA-tagged ATP11A and ATP11C, and parental cells (−) were treated with vehicle alone (white bars), 400 nM of PMA (black bars), or 400 nM of PMA and 2 μM of BIM-1, simultaneously (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated with NBD-PS at 15 °C for 5 min. After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-PS uptake is shown relative to that in Mock-treated parental HeLa cells (−). Graph displays averages from four independent experiments ± SD. *** p
    Figure Legend Snippet: ATP11C is endocytosed by treatment with phorbol 12-myristate 13-acetate (PMA) and increasing cytosolic Ca 2+ . a HeLa cells stably expressing C-terminally HA-tagged ATP11A, and ATP11C were treated for 15 min at 37 °C with vehicle alone (Mock); with either 400 nM of PMA (PMA) or PMA and 2 μM of BIM-1 (PMA + BIM); or with 1 μM A23187 in the presence of either 1.8 mM of CaCl 2 (A23187) or 1.8 mM CaCl 2 and 2 μM BIM-1 (A23187 + BIM). The cells were fixed and immunostained with anti-HA antibody, followed by Cy3-conjugated anti-rat secondary antibody. See Supplementary Fig. 1 and Supplementary Movie 1 . b Cell-surface expression levels of ATP11A and ATP11C following treatment with PMA or A23187 and CaCl 2 were analyzed after surface biotinylation. Proteins precipitated with streptavidin-agarose beads were subjected to immunoblot analysis (left panels, biotinylated). 15% of the input of the biotinylation reaction was loaded in each lane (right panels, total lysate). Expression of ATP11A and ATP11C proteins was analyzed by immunoblotting with anti-HA and anti-ATP1A1 (as an internal control) antibodies. c HeLa cells stably expressing HA-tagged ATP11A and ATP11C, and parental cells (−) were treated with vehicle alone (white bars), 400 nM of PMA (black bars), or 400 nM of PMA and 2 μM of BIM-1, simultaneously (gray bars) for 15 min. The cells were then washed with flippase assay buffer and incubated with NBD-PS at 15 °C for 5 min. After extraction with fatty acid-free BSA, the residual fluorescence intensity associated with the cells was determined by flow cytometry. Fold increase of NBD-PS uptake is shown relative to that in Mock-treated parental HeLa cells (−). Graph displays averages from four independent experiments ± SD. *** p

    Techniques Used: Stable Transfection, Expressing, Incubation, Fluorescence, Flow Cytometry, Cytometry

    15) Product Images from "The Pseudomonas aeruginosa lectin LecB causes integrin internalization to facilitate crawling of bacteria underneath host cells"

    Article Title: The Pseudomonas aeruginosa lectin LecB causes integrin internalization to facilitate crawling of bacteria underneath host cells

    Journal: bioRxiv

    doi: 10.1101/2019.12.12.872739

    LecB directly binds to β1-integrin (A) LecB-biotin was applied apically (AP) or basolaterally (BL) to polarized filter grown MDCK cells or cells were mock-treated AP or BL. Cells were lysed and LecB-biotin-receptor complexes were precipitated with streptavidin beads. Afterwards the presence of β1-integrin was probed by Western blot in the precipitate and the remaining supernatant of the precipitation. (B) LecB-Cy3 (red) was applied basolaterally to MDCK cells for 6 h. Cells were fixed and stained for β1-integrin (green). A confocal section (x-y section) crossing the cells in the sub-apical region is displayed, since in this region most internalized vesicles were concentrated. (C) MDCK cells were lysed, β1-integrins were immunoprecipitated and treated or left untreated with PNGase F to remove N-linked glycans. Western blot analysis of the immunoprecipitated β1-integrins was performed and β1-integrin presence was proven by staining with anti-β1-integrin antibodies (white arrows). Also bands from the antibody used for β1-integrin precipitation are visible (white arrowheads) and proteins that putatively co-precipitated with β1-integrin (blue arrows). To probe the binding of LecB to β1-integrin, LecB-Cy5 was incubated with membranes (far Western assay).
    Figure Legend Snippet: LecB directly binds to β1-integrin (A) LecB-biotin was applied apically (AP) or basolaterally (BL) to polarized filter grown MDCK cells or cells were mock-treated AP or BL. Cells were lysed and LecB-biotin-receptor complexes were precipitated with streptavidin beads. Afterwards the presence of β1-integrin was probed by Western blot in the precipitate and the remaining supernatant of the precipitation. (B) LecB-Cy3 (red) was applied basolaterally to MDCK cells for 6 h. Cells were fixed and stained for β1-integrin (green). A confocal section (x-y section) crossing the cells in the sub-apical region is displayed, since in this region most internalized vesicles were concentrated. (C) MDCK cells were lysed, β1-integrins were immunoprecipitated and treated or left untreated with PNGase F to remove N-linked glycans. Western blot analysis of the immunoprecipitated β1-integrins was performed and β1-integrin presence was proven by staining with anti-β1-integrin antibodies (white arrows). Also bands from the antibody used for β1-integrin precipitation are visible (white arrowheads) and proteins that putatively co-precipitated with β1-integrin (blue arrows). To probe the binding of LecB to β1-integrin, LecB-Cy5 was incubated with membranes (far Western assay).

    Techniques Used: Western Blot, Staining, Immunoprecipitation, Binding Assay, Incubation

    16) Product Images from "Sorting nexin 12 interacts with BACE1 and regulates BACE1-mediated APP processing"

    Article Title: Sorting nexin 12 interacts with BACE1 and regulates BACE1-mediated APP processing

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-7-30

    Downregulation of SNX12 accelerates the endocytosis of BACE1 and decreases the cell surface level of BACE1. ( A ) After RNAi downregulation of SNX12, SH-SY5Y cells were subjected to biotinylation. Cell lysates were affinity-precipitated with streptavidin-agarose beads to pull down biotinylated proteins that were at the cell surface. The levels of biotinylated BACE1 and APP, as well as their total protein levels, were analyzed by Western blot. Cell surface levels of BACE1 were quantified by densitometry and normalized to that of control (set as one arbitrary unit) for comparison. N = 3, *: p
    Figure Legend Snippet: Downregulation of SNX12 accelerates the endocytosis of BACE1 and decreases the cell surface level of BACE1. ( A ) After RNAi downregulation of SNX12, SH-SY5Y cells were subjected to biotinylation. Cell lysates were affinity-precipitated with streptavidin-agarose beads to pull down biotinylated proteins that were at the cell surface. The levels of biotinylated BACE1 and APP, as well as their total protein levels, were analyzed by Western blot. Cell surface levels of BACE1 were quantified by densitometry and normalized to that of control (set as one arbitrary unit) for comparison. N = 3, *: p

    Techniques Used: Western Blot

    17) Product Images from "Proangiogenic functions of an RGD-SLAY-containing osteopontin icosamer peptide in HUVECs and in the postischemic brain"

    Article Title: Proangiogenic functions of an RGD-SLAY-containing osteopontin icosamer peptide in HUVECs and in the postischemic brain

    Journal: Experimental & Molecular Medicine

    doi: 10.1038/emm.2017.241

    Endogenous α v β 3 -integrin mediates the proangiogenic effect of 20-amino-acid OPN peptide (OPNpt20) in human umbilical vein endothelial cells (HUVECs). ( a , b ) Protein levels of matrix metallopeptidase 9 (MMP9) and vascular endothelial growth factor (VEGF) in conditioned media or HUVEC lysates were assessed by immunoblotting after treatment with OPNpt20 (1 μ M ) or OPNpt20-Db (mutant OPN peptide with both RGD and SLAY replaced; 1 μ M ) for 12 h in the presence or absence of anti-α v β 3 (0.1 μg ml −1 ) antibody or IgG (0.1 μg ml −1 ). The results are presented as the mean±s.e.m. ( n =3) ( b ). ( c ) Direct binding between endogenous integrin α v and biotinylated-OPNpt20 or biotinylated-OPNpt20-RAA was examined using a pull-down assay. HUVEC lysates (2.5 μg μl −1 ) were preincubated with anti-α v β 3 or IgG (0.5 ng μl −1 ) for 15 min and then incubated with biotinylated-OPNpt20 or biotinylated-OPNpt20-RAA for 30 min. Complexes were pulled down with streptavidin beads, and α v -integrin was measured by immunoblot using anti-α v -integrin antibody. Representative images are presented and results are presented as the mean±s.e.m. ( n =4) ( c ). * P
    Figure Legend Snippet: Endogenous α v β 3 -integrin mediates the proangiogenic effect of 20-amino-acid OPN peptide (OPNpt20) in human umbilical vein endothelial cells (HUVECs). ( a , b ) Protein levels of matrix metallopeptidase 9 (MMP9) and vascular endothelial growth factor (VEGF) in conditioned media or HUVEC lysates were assessed by immunoblotting after treatment with OPNpt20 (1 μ M ) or OPNpt20-Db (mutant OPN peptide with both RGD and SLAY replaced; 1 μ M ) for 12 h in the presence or absence of anti-α v β 3 (0.1 μg ml −1 ) antibody or IgG (0.1 μg ml −1 ). The results are presented as the mean±s.e.m. ( n =3) ( b ). ( c ) Direct binding between endogenous integrin α v and biotinylated-OPNpt20 or biotinylated-OPNpt20-RAA was examined using a pull-down assay. HUVEC lysates (2.5 μg μl −1 ) were preincubated with anti-α v β 3 or IgG (0.5 ng μl −1 ) for 15 min and then incubated with biotinylated-OPNpt20 or biotinylated-OPNpt20-RAA for 30 min. Complexes were pulled down with streptavidin beads, and α v -integrin was measured by immunoblot using anti-α v -integrin antibody. Representative images are presented and results are presented as the mean±s.e.m. ( n =4) ( c ). * P

    Techniques Used: Mutagenesis, Binding Assay, Pull Down Assay, Incubation

    18) Product Images from "ATAD3A oligomerization causes neurodegeneration by coupling mitochondrial fragmentation and bioenergetics defects"

    Article Title: ATAD3A oligomerization causes neurodegeneration by coupling mitochondrial fragmentation and bioenergetics defects

    Journal: Nature Communications

    doi: 10.1038/s41467-019-09291-x

    DA1 treatment reduces mitochondrial damage and cell death in HD cell cultures. HdhQ7 and HdhQ111 striatal cells were treated with control TAT or peptide DA1 (1 µM/day for 4 days). a Mitochondrial morphology was determined by staining cells with anti-Tom20 antibody. Scale bar: 10 µm. The percentage of cells with fragmented mitochondria relative to the total number of cells was quantitated. Three independent studies, one-way ANOVA with Tukey’s post-hoc test. b TFAM and PGC1α protein levels were analyzed by WB. Histogram: the relative density of TFAM and PGC1α to Actin. Three independent studies, one-way ANOVA with Tukey’s post-hoc test. c HdhQ7 and HdhQ111 cells were transfected with control siRNA (siCon) or ATAD3A siRNA (siAD) followed by treatment with TAT or DA1 (1 µM/day for 4 days). TFAM and ATAD3A protein levels were analyzed by WB. Histogram: the relative density of TFAM to Actin. Four independent studies, one-way ANOVA with Tukey’s post-hoc test. d mtDNA-encoded or nuclear-encoded mitochondrial electron transport proteins were analyzed by WB with the indicated antibodies. Actin: a loading control. VDAC: a mitochondrial loading control. Histogram: the quantitation of relative mtCO2 protein level to Actin. Three independent studies, one-way ANOVA with Tukey’s post-hoc test. e TFAM and mtDNA LSP interaction was analyzed by biotin-streptavidin pull down. Histogram: the relative density of TFAM in the Biotin-LSP precipitates. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. f mtDNA lesion was measured by qPCR. Representative DNA gel is showed in Supplementary Fig. 7J . Five independent studies, one-way ANOVA with Tukey’s post-hoc test. g mitoROS was evaluated by mitoSOX fluorescent probe. At least 100 cells per group were analyzed. At least 3 independent studies, one-way ANOVA with Tukey’s post-hoc test. h Mitochondrial respiratory activity was determined by a seahorse analyzer. Three independent studies, one-way ANOVA with Tukey’s post-hoc test. All data are mean ± SEM
    Figure Legend Snippet: DA1 treatment reduces mitochondrial damage and cell death in HD cell cultures. HdhQ7 and HdhQ111 striatal cells were treated with control TAT or peptide DA1 (1 µM/day for 4 days). a Mitochondrial morphology was determined by staining cells with anti-Tom20 antibody. Scale bar: 10 µm. The percentage of cells with fragmented mitochondria relative to the total number of cells was quantitated. Three independent studies, one-way ANOVA with Tukey’s post-hoc test. b TFAM and PGC1α protein levels were analyzed by WB. Histogram: the relative density of TFAM and PGC1α to Actin. Three independent studies, one-way ANOVA with Tukey’s post-hoc test. c HdhQ7 and HdhQ111 cells were transfected with control siRNA (siCon) or ATAD3A siRNA (siAD) followed by treatment with TAT or DA1 (1 µM/day for 4 days). TFAM and ATAD3A protein levels were analyzed by WB. Histogram: the relative density of TFAM to Actin. Four independent studies, one-way ANOVA with Tukey’s post-hoc test. d mtDNA-encoded or nuclear-encoded mitochondrial electron transport proteins were analyzed by WB with the indicated antibodies. Actin: a loading control. VDAC: a mitochondrial loading control. Histogram: the quantitation of relative mtCO2 protein level to Actin. Three independent studies, one-way ANOVA with Tukey’s post-hoc test. e TFAM and mtDNA LSP interaction was analyzed by biotin-streptavidin pull down. Histogram: the relative density of TFAM in the Biotin-LSP precipitates. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. f mtDNA lesion was measured by qPCR. Representative DNA gel is showed in Supplementary Fig. 7J . Five independent studies, one-way ANOVA with Tukey’s post-hoc test. g mitoROS was evaluated by mitoSOX fluorescent probe. At least 100 cells per group were analyzed. At least 3 independent studies, one-way ANOVA with Tukey’s post-hoc test. h Mitochondrial respiratory activity was determined by a seahorse analyzer. Three independent studies, one-way ANOVA with Tukey’s post-hoc test. All data are mean ± SEM

    Techniques Used: Staining, Western Blot, Transfection, Quantitation Assay, Real-time Polymerase Chain Reaction, Activity Assay

    ATAD3A deacetylation. a Total cell lysates of HdhQ7 and HdhQ111 cells or fibroblasts of HD patient and control subjects were subject to IP with anti-ATAD3A antibodies followed by WB with anti-acetyl lysine antibodies. Asterisks (*) indicates the acetylated ATAD3A. b ATAD3A K135 in peptide 130-AQYADLLAR-138 in human (K134 in mouse) was identified as an acetylated site (also see Supplementary Fig. 6A ). The K135 labeled in pink is present on the surface of ATAD3A N-terminus simulated structure. HEK293 cells were transfected with the indicated Flag-tagged K135 mutants or ATAD3A WT for 48 h. c ATAD3A dimers were analyzed by WB in the presence or absence of β-ME. Histogram: the quantification of relative density of ATAD3A dimer in the absence of β-ME versus total protein level under reduced conditions. At least 6 independent experiments, one-way ANOVA with Tukey’s post-hoc test. d Immunoprecipiates with anti-Flag antibodies were analyzed with anti-Myc antibodies. Histogram: the quantification of relative density of Myc-ATAD3A/ATAD3A-Flag in the immunoprecipitates. Five independent experiments, one-way ANOVA with Tukey’s post-hoc test. e IP of total protein lysates was performed with the indicated antibodies. f HeLa cells were transfected with Flag-ATAD3A WT or K mutants for 48 h. Cells were stained with anti-Tom20 (green) and anti-Flag (red) antibodies. Mitochondrial morphology was imaged. Scale bar: 10 µm. Histogram: the percentage of cells with fragmented mitochondria to total number of cells. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. g The binding of TFAM and biotinylated mtDNA LSP probe was determined by biotin-streptavidin pull down. Histogram: the relative density of TFAM in the Biotin-LSP precipitates. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. h HeLa cells were stained with anti-Tom20 (red) and anti-DNA (blue) antibodies at the indicated groups. The co-localization of DNA and Tom20 was analyzed by confocal microscopy. Insert: the enlarged images. Scale bar: 10 µm. The number of nucleoids immunopositive for both anti-DNA and anti-Tom20 was quantitated by NIH Image J software. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. All shown blots are from at least 3 independent experiments. Data are mean ± SEM
    Figure Legend Snippet: ATAD3A deacetylation. a Total cell lysates of HdhQ7 and HdhQ111 cells or fibroblasts of HD patient and control subjects were subject to IP with anti-ATAD3A antibodies followed by WB with anti-acetyl lysine antibodies. Asterisks (*) indicates the acetylated ATAD3A. b ATAD3A K135 in peptide 130-AQYADLLAR-138 in human (K134 in mouse) was identified as an acetylated site (also see Supplementary Fig. 6A ). The K135 labeled in pink is present on the surface of ATAD3A N-terminus simulated structure. HEK293 cells were transfected with the indicated Flag-tagged K135 mutants or ATAD3A WT for 48 h. c ATAD3A dimers were analyzed by WB in the presence or absence of β-ME. Histogram: the quantification of relative density of ATAD3A dimer in the absence of β-ME versus total protein level under reduced conditions. At least 6 independent experiments, one-way ANOVA with Tukey’s post-hoc test. d Immunoprecipiates with anti-Flag antibodies were analyzed with anti-Myc antibodies. Histogram: the quantification of relative density of Myc-ATAD3A/ATAD3A-Flag in the immunoprecipitates. Five independent experiments, one-way ANOVA with Tukey’s post-hoc test. e IP of total protein lysates was performed with the indicated antibodies. f HeLa cells were transfected with Flag-ATAD3A WT or K mutants for 48 h. Cells were stained with anti-Tom20 (green) and anti-Flag (red) antibodies. Mitochondrial morphology was imaged. Scale bar: 10 µm. Histogram: the percentage of cells with fragmented mitochondria to total number of cells. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. g The binding of TFAM and biotinylated mtDNA LSP probe was determined by biotin-streptavidin pull down. Histogram: the relative density of TFAM in the Biotin-LSP precipitates. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. h HeLa cells were stained with anti-Tom20 (red) and anti-DNA (blue) antibodies at the indicated groups. The co-localization of DNA and Tom20 was analyzed by confocal microscopy. Insert: the enlarged images. Scale bar: 10 µm. The number of nucleoids immunopositive for both anti-DNA and anti-Tom20 was quantitated by NIH Image J software. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. All shown blots are from at least 3 independent experiments. Data are mean ± SEM

    Techniques Used: Western Blot, Labeling, Transfection, Staining, Binding Assay, Confocal Microscopy, Software

    ATAD3A oligomerization impairs mitochondrial fission and mtDNA stability. HdhQ7 and Q111 cells were transfected with control siRNA (NC) or ATAD3A siRNA (siA) for 3 days. a Downregulation of ATAD3A was validated by WB. Actin: a loading control. Drp1 polymerization was analyzed by WB with anti-Drp1 antibodies in the absence of β-ME. b Mitochondria were isolated from HD striatal cells (Upper) and cells exposed to 5 mM 3-NP for 4 h (Lower). Drp1 mitochondrial level was analyzed by WB. Mitochondrial loading control: VDAC. c HeLa cells were transfected with ATAD3A-GFP truncated mutants, shown in Supplementary Fig. 3E , for 48 h. Cells were stained with anti-Tom20 (red) and anti-DNA (cyan) antibodies. The co-localization of DNA and Tom20 was analyzed by confocal microscopy. Insert: the enlarged images. Scale bar: 10 µm. d The number of nucleoids immunopositive for both anti-DNA and anti-Tom20 was quantitated by NIH Image J software. 40–50 cells per group were analyzed. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. e Neuro2A cells were transfected with ATAD3A-Flag WT or mutants for 48 h, and total DNA was extracted for qPCR analysis to monitor the mtDNA lesion. Upper: Representative DNA agarose gel of the amplification of the 10 kb mtDNA fragment. Lower: The quantification of mtDNA lesion. Four independent experiments, one-way ANOVA with Tukey’s post-hoc test. f mtDNA content was analyzed by qPCR using primers from D-loop (left). TFAM mRNA level was analyzed by qPCR (right). At least 3 independent experiments, one-way ANOVA with Tukey’s post-hoc test. g Left: Cells were stained with mitoSOX fluorescent probe to evaluate mitochondrial superoxide production (mitoROS). Right: Cell death rate was measured by LDH release into cytosol. Four independent experiments, one-way ANOVA with Tukey’s post-hoc test. h The binding of TFAM and biotinylated mtDNA LSP probe was determined by biotin-streptavidin pull down in HD striatal cells transfected with ATAD3A siRNA (left) and in Neuo2A cells expressing ATAD3A truncated mutants (right). All shown representative blots are from at least 3 independent experiments. All data are mean ± SEM
    Figure Legend Snippet: ATAD3A oligomerization impairs mitochondrial fission and mtDNA stability. HdhQ7 and Q111 cells were transfected with control siRNA (NC) or ATAD3A siRNA (siA) for 3 days. a Downregulation of ATAD3A was validated by WB. Actin: a loading control. Drp1 polymerization was analyzed by WB with anti-Drp1 antibodies in the absence of β-ME. b Mitochondria were isolated from HD striatal cells (Upper) and cells exposed to 5 mM 3-NP for 4 h (Lower). Drp1 mitochondrial level was analyzed by WB. Mitochondrial loading control: VDAC. c HeLa cells were transfected with ATAD3A-GFP truncated mutants, shown in Supplementary Fig. 3E , for 48 h. Cells were stained with anti-Tom20 (red) and anti-DNA (cyan) antibodies. The co-localization of DNA and Tom20 was analyzed by confocal microscopy. Insert: the enlarged images. Scale bar: 10 µm. d The number of nucleoids immunopositive for both anti-DNA and anti-Tom20 was quantitated by NIH Image J software. 40–50 cells per group were analyzed. Three independent experiments, one-way ANOVA with Tukey’s post-hoc test. e Neuro2A cells were transfected with ATAD3A-Flag WT or mutants for 48 h, and total DNA was extracted for qPCR analysis to monitor the mtDNA lesion. Upper: Representative DNA agarose gel of the amplification of the 10 kb mtDNA fragment. Lower: The quantification of mtDNA lesion. Four independent experiments, one-way ANOVA with Tukey’s post-hoc test. f mtDNA content was analyzed by qPCR using primers from D-loop (left). TFAM mRNA level was analyzed by qPCR (right). At least 3 independent experiments, one-way ANOVA with Tukey’s post-hoc test. g Left: Cells were stained with mitoSOX fluorescent probe to evaluate mitochondrial superoxide production (mitoROS). Right: Cell death rate was measured by LDH release into cytosol. Four independent experiments, one-way ANOVA with Tukey’s post-hoc test. h The binding of TFAM and biotinylated mtDNA LSP probe was determined by biotin-streptavidin pull down in HD striatal cells transfected with ATAD3A siRNA (left) and in Neuo2A cells expressing ATAD3A truncated mutants (right). All shown representative blots are from at least 3 independent experiments. All data are mean ± SEM

    Techniques Used: Transfection, Western Blot, Isolation, Staining, Confocal Microscopy, Software, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Binding Assay, Expressing

    19) Product Images from "The Pseudomonas aeruginosa lectin LecB causes integrin internalization to facilitate crawling of bacteria underneath host cells"

    Article Title: The Pseudomonas aeruginosa lectin LecB causes integrin internalization to facilitate crawling of bacteria underneath host cells

    Journal: bioRxiv

    doi: 10.1101/2019.12.12.872739

    LecB directly binds to β1-integrin (A) LecB-biotin was applied apically (AP) or basolaterally (BL) to polarized filter grown MDCK cells or cells were mock-treated AP or BL. Cells were lysed and LecB-biotin-receptor complexes were precipitated with streptavidin beads. Afterwards the presence of β1-integrin was probed by Western blot in the precipitate and the remaining supernatant of the precipitation. (B) LecB-Cy3 (red) was applied basolaterally to MDCK cells for 6 h. Cells were fixed and stained for β1-integrin (green). A confocal section (x-y section) crossing the cells in the sub-apical region is displayed, since in this region most internalized vesicles were concentrated. (C) MDCK cells were lysed, β1-integrins were immunoprecipitated and treated or left untreated with PNGase F to remove N-linked glycans. Western blot analysis of the immunoprecipitated β1-integrins was performed and β1-integrin presence was proven by staining with anti-β1-integrin antibodies (white arrows). Also bands from the antibody used for β1-integrin precipitation are visible (white arrowheads) and proteins that putatively co-precipitated with β1-integrin (blue arrows). To probe the binding of LecB to β1-integrin, LecB-Cy5 was incubated with membranes (far Western assay).
    Figure Legend Snippet: LecB directly binds to β1-integrin (A) LecB-biotin was applied apically (AP) or basolaterally (BL) to polarized filter grown MDCK cells or cells were mock-treated AP or BL. Cells were lysed and LecB-biotin-receptor complexes were precipitated with streptavidin beads. Afterwards the presence of β1-integrin was probed by Western blot in the precipitate and the remaining supernatant of the precipitation. (B) LecB-Cy3 (red) was applied basolaterally to MDCK cells for 6 h. Cells were fixed and stained for β1-integrin (green). A confocal section (x-y section) crossing the cells in the sub-apical region is displayed, since in this region most internalized vesicles were concentrated. (C) MDCK cells were lysed, β1-integrins were immunoprecipitated and treated or left untreated with PNGase F to remove N-linked glycans. Western blot analysis of the immunoprecipitated β1-integrins was performed and β1-integrin presence was proven by staining with anti-β1-integrin antibodies (white arrows). Also bands from the antibody used for β1-integrin precipitation are visible (white arrowheads) and proteins that putatively co-precipitated with β1-integrin (blue arrows). To probe the binding of LecB to β1-integrin, LecB-Cy5 was incubated with membranes (far Western assay).

    Techniques Used: Western Blot, Staining, Immunoprecipitation, Binding Assay, Incubation

    20) Product Images from "How Stat1 mediates constitutive gene expression: a complex of unphosphorylated Stat1 and IRF1 supports transcription of the LMP2 gene"

    Article Title: How Stat1 mediates constitutive gene expression: a complex of unphosphorylated Stat1 and IRF1 supports transcription of the LMP2 gene

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.15.4111

    Fig. 8. A complex of Stat1 and IRF1 binds to the LMP2 GAS or LMP2 1/2 GAS. Streptavidin–agarose beads were saturated with biotin-labeled oligonucleotides at 4°C for 2 h. The beads were incubated with either Stat1 (2–5 µg), IRF1 ( in vitro translation products), U3A extracts or U3A-IRF1(H) extracts at 4°C for 1 h. The Stat1-saturated beads were then washed with HEM buffer and incubated with either IRF1 ( in vitro translation product), U3A extracts or U3A-IRF1(H) extracts. Samples were analyzed in western transfers with anti-IRF1.
    Figure Legend Snippet: Fig. 8. A complex of Stat1 and IRF1 binds to the LMP2 GAS or LMP2 1/2 GAS. Streptavidin–agarose beads were saturated with biotin-labeled oligonucleotides at 4°C for 2 h. The beads were incubated with either Stat1 (2–5 µg), IRF1 ( in vitro translation products), U3A extracts or U3A-IRF1(H) extracts at 4°C for 1 h. The Stat1-saturated beads were then washed with HEM buffer and incubated with either IRF1 ( in vitro translation product), U3A extracts or U3A-IRF1(H) extracts. Samples were analyzed in western transfers with anti-IRF1.

    Techniques Used: Labeling, Incubation, In Vitro, Western Blot

    21) Product Images from "P113 is a merozoite surface protein that binds the N terminus of Plasmodium falciparum RH5"

    Article Title: P113 is a merozoite surface protein that binds the N terminus of Plasmodium falciparum RH5

    Journal: Nature Communications

    doi: 10.1038/ncomms14333

    The interaction between P113 and the N-terminal region of RH5 is specific and 10-fold stronger in the context of full-length RH5. ( a ) RH5Nt bound P113 specifically and with low affinity. Purified monomeric RH5Nt was serially diluted before being injected over 780 RU of monobiotinylated P113 immobilized on a streptavidin-coated sensor chip. The binding traces suggested the presence of a small amount of multimeric material (see Methods); consequently, binding equilibrium was not quite achieved even though long ( > 60 s) injection times were used; the dashed line (inset) marks the time point used for equilibrium binding. From these data, an equilibrium binding constant ( K D ) of 3.0±0.5 μM (mean±s.d., n =2) was calculated. ( b , c ) RH5FL binds P113 with a ten-fold higher affinity than RH5Nt. A kinetic analysis of P113 binding RH5Nt ( b ) or RH5FL ( c ) using SPR; inset gels demonstrate the homogeneity and integrity of each RH5 analyte preparation. Serially diluted RH5 analytes were injected over 780 RU of immobilized P113. Binding data (black lines) fitted a simple 1:1 binding model well (red lines), and were used to determine binding constants. ( d ) Gel filtration elution profiles of the entire ectodomain (EE) and an N-terminal subfragment (Y1-N653) of recombinant tagged P113 demonstrated that P113 EE can form multimers. The lettered dashed lines correspond to fractions collected over the resolved peaks; the elution volumes of gel filtration mass markers are indicated. ( e ) Native and denaturing (SDS–PAGE) gels corresponding to aliquots of the fractions indicated by the letters in d . P113 EE but not the Y1-N653 fragment resolves at an approximately fourfold higher mass under native when compared with denaturing conditions. The Y1-N653 fragment resolved as two species by native PAGE. Multimeric forms of P113 EE exhibited complex multivalent binding behaviour by SPR ( f ) compared with P113 Y1-N653 ( g ). Sensorgrams in ( f , g ) show a 120 s pulse of purified 8 μM P113 analyte injected over 510 RU of immobilized RH5Nt.
    Figure Legend Snippet: The interaction between P113 and the N-terminal region of RH5 is specific and 10-fold stronger in the context of full-length RH5. ( a ) RH5Nt bound P113 specifically and with low affinity. Purified monomeric RH5Nt was serially diluted before being injected over 780 RU of monobiotinylated P113 immobilized on a streptavidin-coated sensor chip. The binding traces suggested the presence of a small amount of multimeric material (see Methods); consequently, binding equilibrium was not quite achieved even though long ( > 60 s) injection times were used; the dashed line (inset) marks the time point used for equilibrium binding. From these data, an equilibrium binding constant ( K D ) of 3.0±0.5 μM (mean±s.d., n =2) was calculated. ( b , c ) RH5FL binds P113 with a ten-fold higher affinity than RH5Nt. A kinetic analysis of P113 binding RH5Nt ( b ) or RH5FL ( c ) using SPR; inset gels demonstrate the homogeneity and integrity of each RH5 analyte preparation. Serially diluted RH5 analytes were injected over 780 RU of immobilized P113. Binding data (black lines) fitted a simple 1:1 binding model well (red lines), and were used to determine binding constants. ( d ) Gel filtration elution profiles of the entire ectodomain (EE) and an N-terminal subfragment (Y1-N653) of recombinant tagged P113 demonstrated that P113 EE can form multimers. The lettered dashed lines correspond to fractions collected over the resolved peaks; the elution volumes of gel filtration mass markers are indicated. ( e ) Native and denaturing (SDS–PAGE) gels corresponding to aliquots of the fractions indicated by the letters in d . P113 EE but not the Y1-N653 fragment resolves at an approximately fourfold higher mass under native when compared with denaturing conditions. The Y1-N653 fragment resolved as two species by native PAGE. Multimeric forms of P113 EE exhibited complex multivalent binding behaviour by SPR ( f ) compared with P113 Y1-N653 ( g ). Sensorgrams in ( f , g ) show a 120 s pulse of purified 8 μM P113 analyte injected over 510 RU of immobilized RH5Nt.

    Techniques Used: Affinity Purification, Injection, Chromatin Immunoprecipitation, Binding Assay, SPR Assay, Filtration, Recombinant, SDS Page, Clear Native PAGE, Purification

    An ‘amph-vaccine' based on RH5Nt elicits antibodies that inhibit parasite growth in vitro . ( a ) A synthetic 116 amino-acid peptide corresponding to RH5Nt interacts with P113. Serial dilutions of purified P113 (Y1-N653) were injected over the RH5Nt peptide immobilized on a streptavidin-coated sensor chip after the C-terminal cysteine was conjugated to biotin functionalized with maleimide. Reference-subtracted binding data are shown (black lines) which fit well to a 1:1 binding model (red lines). ( b ) Structure of an ‘amph-vaccine' based on RH5Nt created by conjugating the RH5Nt peptide to maleimide-functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N -[maleimide(polyethylene glycol)-2000] (PEG 2000 -DSPE). Antibodies raised against RH5Nt peptide (Anti-pRH5Nt) blocked RH5 binding to P113 ( c ), but not to basigin ( d ). The indicated concentrations of protein-G purified rabbit polyclonal antibodies were incubated with RH5 β-lactamase-tagged prey proteins before presenting them to immobilized P113 ( c ) or basigin ( d ) baits. Prey binding was quantified by nitrocefin hydrolysis at 485 nm; polyclonal antibodies to RH5FL and AMA1 were used as positive and negative controls respectively. ( e ) Polyclonal antibodies elicited against the RH5Nt amph-vaccine inhibited erythrocyte invasion of both 3D7 and Dd2 strain of P. falciparum . Data points represent means±95% confidence interval, n= 3; a representative experiment from two independent experiments is shown.
    Figure Legend Snippet: An ‘amph-vaccine' based on RH5Nt elicits antibodies that inhibit parasite growth in vitro . ( a ) A synthetic 116 amino-acid peptide corresponding to RH5Nt interacts with P113. Serial dilutions of purified P113 (Y1-N653) were injected over the RH5Nt peptide immobilized on a streptavidin-coated sensor chip after the C-terminal cysteine was conjugated to biotin functionalized with maleimide. Reference-subtracted binding data are shown (black lines) which fit well to a 1:1 binding model (red lines). ( b ) Structure of an ‘amph-vaccine' based on RH5Nt created by conjugating the RH5Nt peptide to maleimide-functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- N -[maleimide(polyethylene glycol)-2000] (PEG 2000 -DSPE). Antibodies raised against RH5Nt peptide (Anti-pRH5Nt) blocked RH5 binding to P113 ( c ), but not to basigin ( d ). The indicated concentrations of protein-G purified rabbit polyclonal antibodies were incubated with RH5 β-lactamase-tagged prey proteins before presenting them to immobilized P113 ( c ) or basigin ( d ) baits. Prey binding was quantified by nitrocefin hydrolysis at 485 nm; polyclonal antibodies to RH5FL and AMA1 were used as positive and negative controls respectively. ( e ) Polyclonal antibodies elicited against the RH5Nt amph-vaccine inhibited erythrocyte invasion of both 3D7 and Dd2 strain of P. falciparum . Data points represent means±95% confidence interval, n= 3; a representative experiment from two independent experiments is shown.

    Techniques Used: In Vitro, Purification, Injection, Chromatin Immunoprecipitation, Binding Assay, Incubation

    The N-terminal domain of RH5 is not involved in basigin binding. ( a ) Purified tagged recombinant RH5 expressed by HEK293 cells resolved as four species by SDS–PAGE under reducing conditions. The N-terminal sequence of each species was determined by Edman degradation and is shown. ( b ) The N-terminal region of RH5 is not involved in basigin binding. The indicated RH5 fragments were expressed as biotinylated bait proteins and probed for interactions with a highly avid β-lactamase-tagged basigin prey protein using the AVEXIS assay. The full length (FL) and the major processed form (RH5Ct) of RH5 bound basigin but the N-terminal region (RH5Nt) did not. Bars represent means±95% confidence intervals; n =3. ( c ) Biophysical analysis of the RH5Ct-basigin interaction. Serial dilutions of the indicated concentrations of purified RH5Ct protein were injected over the biotinylated ectodomain of basigin immobilized on a streptavidin-coated sensor chip. The data showed excellent fits to a simple (1:1) binding model (red line). The binding parameters of RH5Ct did not differ significantly from those obtained from RH5FL ( Supplementary Table 1 ) demonstrating that the N-terminal region of RH5 does not contribute to basigin binding affinity.
    Figure Legend Snippet: The N-terminal domain of RH5 is not involved in basigin binding. ( a ) Purified tagged recombinant RH5 expressed by HEK293 cells resolved as four species by SDS–PAGE under reducing conditions. The N-terminal sequence of each species was determined by Edman degradation and is shown. ( b ) The N-terminal region of RH5 is not involved in basigin binding. The indicated RH5 fragments were expressed as biotinylated bait proteins and probed for interactions with a highly avid β-lactamase-tagged basigin prey protein using the AVEXIS assay. The full length (FL) and the major processed form (RH5Ct) of RH5 bound basigin but the N-terminal region (RH5Nt) did not. Bars represent means±95% confidence intervals; n =3. ( c ) Biophysical analysis of the RH5Ct-basigin interaction. Serial dilutions of the indicated concentrations of purified RH5Ct protein were injected over the biotinylated ectodomain of basigin immobilized on a streptavidin-coated sensor chip. The data showed excellent fits to a simple (1:1) binding model (red line). The binding parameters of RH5Ct did not differ significantly from those obtained from RH5FL ( Supplementary Table 1 ) demonstrating that the N-terminal region of RH5 does not contribute to basigin binding affinity.

    Techniques Used: Binding Assay, Purification, Recombinant, SDS Page, Sequencing, Injection, Chromatin Immunoprecipitation

    22) Product Images from "BACE1 Protein Endocytosis and Trafficking Are Differentially Regulated by Ubiquitination at Lysine 501 and the Di-leucine Motif in the Carboxyl Terminus *"

    Article Title: BACE1 Protein Endocytosis and Trafficking Are Differentially Regulated by Ubiquitination at Lysine 501 and the Di-leucine Motif in the Carboxyl Terminus *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.407072

    Carboxyl-terminal di-leucine motif but not ubiquitination at Lys-501 regulates BACE1 endocytosis. A, rate of BACE1 endocytosis analyzed via cell-surface biotinylation and pulldown with streptavidin beads. Levels of BACE1 were analyzed by WB using anti-V5
    Figure Legend Snippet: Carboxyl-terminal di-leucine motif but not ubiquitination at Lys-501 regulates BACE1 endocytosis. A, rate of BACE1 endocytosis analyzed via cell-surface biotinylation and pulldown with streptavidin beads. Levels of BACE1 were analyzed by WB using anti-V5

    Techniques Used: Western Blot

    23) Product Images from "Soybean ENOD40 encodes two peptides that bind to sucrose synthase"

    Article Title: Soybean ENOD40 encodes two peptides that bind to sucrose synthase

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.022664799

    Peptide A and peptide B bind to the same target protein in nodule extracts. Analysis by SDS/PAGE. Biotinylated peptides A and B (lanes 1 and 3) and the corresponding biotinylated control peptides (lanes 2 and 4) were incubated with extracts from soybean nodules and subsequently bound to streptavidin-agarose beads. Proteins isolated with the peptide affinity matrix were eluted, analyzed by SDS/10% PAGE, and stained with SYPRO ruby. The arrowhead marks the position of the 93-kDa peptide-binding protein. Sizes of molecular mass markers (lane M) are indicated.
    Figure Legend Snippet: Peptide A and peptide B bind to the same target protein in nodule extracts. Analysis by SDS/PAGE. Biotinylated peptides A and B (lanes 1 and 3) and the corresponding biotinylated control peptides (lanes 2 and 4) were incubated with extracts from soybean nodules and subsequently bound to streptavidin-agarose beads. Proteins isolated with the peptide affinity matrix were eluted, analyzed by SDS/10% PAGE, and stained with SYPRO ruby. The arrowhead marks the position of the 93-kDa peptide-binding protein. Sizes of molecular mass markers (lane M) are indicated.

    Techniques Used: SDS Page, Incubation, Isolation, Polyacrylamide Gel Electrophoresis, Staining, Binding Assay

    Binding of wild-type and mutant GmENOD40 peptides A and B to SuSy. ( A ) SuSy was partially purified from soybean nodule extract by gel chromatography on Superdex 200. The SuSy-containing fraction used for binding reactions (bar) and the elution position of marker proteins ferritin, catalase, lactate dehydrogenase, and lysozyme are indicated (arrowheads). ( B and C ) Binding of biotinylated peptides and mutant derivatives to SuSy. Substitutions of amino acid residues in the corresponding peptides are underlined. SuSy was isolated from the Superdex fraction (lane 1) with peptide-streptavidin-agarose beads. Proteins bound to the affinity matrix were eluted by boiling in sample buffer, resolved by SDS/10% PAGE, and stained with SYPRO ruby. Lane M, molecular size markers. The authenticity of the SuSy protein band was verified by Western blotting with anti-SuSy Abs (data not shown).
    Figure Legend Snippet: Binding of wild-type and mutant GmENOD40 peptides A and B to SuSy. ( A ) SuSy was partially purified from soybean nodule extract by gel chromatography on Superdex 200. The SuSy-containing fraction used for binding reactions (bar) and the elution position of marker proteins ferritin, catalase, lactate dehydrogenase, and lysozyme are indicated (arrowheads). ( B and C ) Binding of biotinylated peptides and mutant derivatives to SuSy. Substitutions of amino acid residues in the corresponding peptides are underlined. SuSy was isolated from the Superdex fraction (lane 1) with peptide-streptavidin-agarose beads. Proteins bound to the affinity matrix were eluted by boiling in sample buffer, resolved by SDS/10% PAGE, and stained with SYPRO ruby. Lane M, molecular size markers. The authenticity of the SuSy protein band was verified by Western blotting with anti-SuSy Abs (data not shown).

    Techniques Used: Binding Assay, Mutagenesis, Purification, Chromatography, Marker, Isolation, Polyacrylamide Gel Electrophoresis, Staining, Western Blot

    24) Product Images from "Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex"

    Article Title: Reinvestigation of Aminoacyl-TRNA Synthetase Core Complex by Affinity Purification-Mass Spectrometry Reveals TARSL2 as a Potential Member of the Complex

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081734

    Affinity purification of SBP-tagged AIMP1, AIMP2 and KARS. ( A ) Schematic diagram of AIMP1, AIMP2, and KARS constructs for affinity purification. S/FLAG/SBP tags were attached to the N-terminus of cloned genes. ( B ) Expression of AIMP1, AIMP2, and KARS tagged with S/FLAG/SBP in HEK 293T cells were confirmed by immunoblotting analysis using anti-FLAG antibody. Closed arrowheads (◀) indicate AIMP1, AIMP2 and KARS. ( C ) Streptavidin affinity purification was carried out and 10 % of the eluted samples from HEK 293T and HCT-8 cells were visualized by protein staining. One of three biological replicates is shown and the bait proteins are marked with red arrows. ( D ) 90 % of elution was separated on SDS-PAGE to about 1-cm distance and divided into three fractions each. Then, tryptic peptides were recovered from each gel bands and analyzed by LC-MS/MS. SAINT algorithm was used to calculate the likelihood of true interaction of identified proteins. M; Mock, A1; AIMP1, A2; AIMP2, K; KARS. ‘Mock’ is a vector having the S/FLAG/SBP tag only without target genes.
    Figure Legend Snippet: Affinity purification of SBP-tagged AIMP1, AIMP2 and KARS. ( A ) Schematic diagram of AIMP1, AIMP2, and KARS constructs for affinity purification. S/FLAG/SBP tags were attached to the N-terminus of cloned genes. ( B ) Expression of AIMP1, AIMP2, and KARS tagged with S/FLAG/SBP in HEK 293T cells were confirmed by immunoblotting analysis using anti-FLAG antibody. Closed arrowheads (◀) indicate AIMP1, AIMP2 and KARS. ( C ) Streptavidin affinity purification was carried out and 10 % of the eluted samples from HEK 293T and HCT-8 cells were visualized by protein staining. One of three biological replicates is shown and the bait proteins are marked with red arrows. ( D ) 90 % of elution was separated on SDS-PAGE to about 1-cm distance and divided into three fractions each. Then, tryptic peptides were recovered from each gel bands and analyzed by LC-MS/MS. SAINT algorithm was used to calculate the likelihood of true interaction of identified proteins. M; Mock, A1; AIMP1, A2; AIMP2, K; KARS. ‘Mock’ is a vector having the S/FLAG/SBP tag only without target genes.

    Techniques Used: Affinity Purification, Construct, Clone Assay, Expressing, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Plasmid Preparation

    TARSL2 as a member of ARS core complex. ( A , B ) TARSL2 and TARS were detected in AIMP1, AIMP2, and KARS immunoprecipitates of HEK 293T ( A ) and HCT-8 cells ( B ). EPRS was used as a positive control. Actin was used for loading control. TCL; total cell lysate, SA pull down; streptavidin pull down. ( C ) Endogenous EPRS was co-immunoprecipitated with TARSL2. Cell lysate (500 μg) was immunoprecipitated with the antibodies against TARSL2, TARS, and IgG and probed for EPRS, TARSL2 and TARS. ( D ) Reciprocal co-immunoprecipitation. Cell lysate (500 μg) was immunoprecipitated with the antibodies against EPRS, AIMP1, AIMP2, KARS and IgG and probed for TARSL2. IgG was used for immunoprecipitation control. M; Mock, E; ERPS, A1; AIMP1, A2; AIMP2, K; KARS, In; Input, TL2; TARSL2, T; TARS, IgG(R); rabbit IgG, IgG(M); mouse IgG. Closed arrowheads (▶) indicate TARSL2 and TARS.
    Figure Legend Snippet: TARSL2 as a member of ARS core complex. ( A , B ) TARSL2 and TARS were detected in AIMP1, AIMP2, and KARS immunoprecipitates of HEK 293T ( A ) and HCT-8 cells ( B ). EPRS was used as a positive control. Actin was used for loading control. TCL; total cell lysate, SA pull down; streptavidin pull down. ( C ) Endogenous EPRS was co-immunoprecipitated with TARSL2. Cell lysate (500 μg) was immunoprecipitated with the antibodies against TARSL2, TARS, and IgG and probed for EPRS, TARSL2 and TARS. ( D ) Reciprocal co-immunoprecipitation. Cell lysate (500 μg) was immunoprecipitated with the antibodies against EPRS, AIMP1, AIMP2, KARS and IgG and probed for TARSL2. IgG was used for immunoprecipitation control. M; Mock, E; ERPS, A1; AIMP1, A2; AIMP2, K; KARS, In; Input, TL2; TARSL2, T; TARS, IgG(R); rabbit IgG, IgG(M); mouse IgG. Closed arrowheads (▶) indicate TARSL2 and TARS.

    Techniques Used: Positive Control, Immunoprecipitation

    25) Product Images from "Adenosinergic signaling inhibits oxalate transport by human intestinal Caco2-BBE cells through the A2B adenosine receptor"

    Article Title: Adenosinergic signaling inhibits oxalate transport by human intestinal Caco2-BBE cells through the A2B adenosine receptor

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00024.2017

    Effect of adenosine (ADO) on surface alkaline phosphatase (AKP) protein expression in Caco2-BBE (C2) cells. A : a representative Western blot analysis of surface AKP protein expression. C2 cells were preincubated with vehicle (Control) or 100 µM ADO for 2 min in the culture medium, and then AKP surface protein expression was evaluated after streptavidin precipitation of surface biotinylated proteins from 300 µg of initial cell lysate. The lower half of the same blot was probed with an anti-GAPDH antibody. B : densitometry of immunoblot results. Western blot band density was quantified using ImageJ software. Values are means ± SE for 3 independent experiments and are presented as a percentage of the respective Control value. ADO has no significant effect on AKP surface protein expression.
    Figure Legend Snippet: Effect of adenosine (ADO) on surface alkaline phosphatase (AKP) protein expression in Caco2-BBE (C2) cells. A : a representative Western blot analysis of surface AKP protein expression. C2 cells were preincubated with vehicle (Control) or 100 µM ADO for 2 min in the culture medium, and then AKP surface protein expression was evaluated after streptavidin precipitation of surface biotinylated proteins from 300 µg of initial cell lysate. The lower half of the same blot was probed with an anti-GAPDH antibody. B : densitometry of immunoblot results. Western blot band density was quantified using ImageJ software. Values are means ± SE for 3 independent experiments and are presented as a percentage of the respective Control value. ADO has no significant effect on AKP surface protein expression.

    Techniques Used: ALP Assay, Expressing, Western Blot, Software

    Effect of adenosine (ADO) on total ( A ) and surface ( B ) PAT1 protein expression in Caco2-BBE (C2) cells. A : a representative Western blot analysis of total PAT1 protein. C2 cells were preincubated with vehicle (CON) or 100 µM ADO for 2 min in the culture medium, and then PAT1 protein expression was evaluated in the cell lysate (30 µg protein/lane). The lower half of the same blot was probed with an anti-GAPDH (GAP) antibody to verify equal loading of protein in each lane (lower panel). B : A representative Western blot analysis of surface PAT1 protein. C2 cells were preincubated with vehicle (CON) or 100 µM ADO for 2 min in the culture medium, and then PAT1 surface protein expression was evaluated after streptavidin precipitation of surface biotinylated proteins from 2,500 µg of initial cell lysate. C : densitometry of immunoblot results. Western blot band density was quantified using ImageJ software. Values are means ± SE for 6 independent experiments (plastic-grown cells) of relative total PAT1 abundance to GAPDH and biotinylated PAT1 and are presented as a percentage of the respective control (CON) value. ADO significantly reduced the amount of PAT1 protein available to surface biotinylation (* P
    Figure Legend Snippet: Effect of adenosine (ADO) on total ( A ) and surface ( B ) PAT1 protein expression in Caco2-BBE (C2) cells. A : a representative Western blot analysis of total PAT1 protein. C2 cells were preincubated with vehicle (CON) or 100 µM ADO for 2 min in the culture medium, and then PAT1 protein expression was evaluated in the cell lysate (30 µg protein/lane). The lower half of the same blot was probed with an anti-GAPDH (GAP) antibody to verify equal loading of protein in each lane (lower panel). B : A representative Western blot analysis of surface PAT1 protein. C2 cells were preincubated with vehicle (CON) or 100 µM ADO for 2 min in the culture medium, and then PAT1 surface protein expression was evaluated after streptavidin precipitation of surface biotinylated proteins from 2,500 µg of initial cell lysate. C : densitometry of immunoblot results. Western blot band density was quantified using ImageJ software. Values are means ± SE for 6 independent experiments (plastic-grown cells) of relative total PAT1 abundance to GAPDH and biotinylated PAT1 and are presented as a percentage of the respective control (CON) value. ADO significantly reduced the amount of PAT1 protein available to surface biotinylation (* P

    Techniques Used: Expressing, Western Blot, Software

    26) Product Images from "Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method: evidence for glutathionylation"

    Article Title: Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method: evidence for glutathionylation

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00350.2016

    SDS-PAGE analysis of dimer formation of the PCFT mutants posttranslationally modified. A : PCFT expressed on the plasma membrane in transient transfectants was labeled with lysine-targeted EZ-Link Sulfo-NHS-LC-Biotin and pulled down with streptavidin beads. DTT-free sample buffer was used to mix the crude membrane fractions at room temperature and to strip proteins from the beads at 100°C for 5 min. The protein samples were then mixed with equal volumes of either DTT-free sample buffer (DTT−) or DTT-containing sample buffer (DTT+) for at least 30 min at room temperature before being loaded on the gel. B : transient transfectants were treated successively with DTT, N -ethylmaleimide (NEM), and EZ-Link Sulfo-NHS-LC-Biotin before biotinylated PCFT was pulled down with streptavidin beads. DTT-containing or DTT-free sample buffer were used to process the crude membranes and the beads. Each panel is a representative image from at least two separate experiments.
    Figure Legend Snippet: SDS-PAGE analysis of dimer formation of the PCFT mutants posttranslationally modified. A : PCFT expressed on the plasma membrane in transient transfectants was labeled with lysine-targeted EZ-Link Sulfo-NHS-LC-Biotin and pulled down with streptavidin beads. DTT-free sample buffer was used to mix the crude membrane fractions at room temperature and to strip proteins from the beads at 100°C for 5 min. The protein samples were then mixed with equal volumes of either DTT-free sample buffer (DTT−) or DTT-containing sample buffer (DTT+) for at least 30 min at room temperature before being loaded on the gel. B : transient transfectants were treated successively with DTT, N -ethylmaleimide (NEM), and EZ-Link Sulfo-NHS-LC-Biotin before biotinylated PCFT was pulled down with streptavidin beads. DTT-containing or DTT-free sample buffer were used to process the crude membranes and the beads. Each panel is a representative image from at least two separate experiments.

    Techniques Used: SDS Page, Modification, Labeling, Stripping Membranes

    Glutathionylation of the substituted cysteine residues in PCFT-mutants. Panel A : Glutathionylation of PCFT mutants that were found to be posttranslationally modified. After transient transfection, cells were grown in RPMI-1640 medium containing 22 µM BioGEE for 24 h before cells were processed for pull-down assay with streptavidin beads. The last lane of this panel serves as a quantitative control in which PCFT expressed on the plasma membrane (P.M.) was labeled with EZ-Link Sulfo-NHS-LC-Biotin. B : the effects of DTT on the removal of the glutathionylation modification from PCFT mutants. Transient transfectants were labeled with BioGEE for 24 h and then treated with DTT (DTT+) or buffer alone (DTT−) before pull-down assays were performed with streptavidin beads. C : the effects of addition of GSSG on biotinylation of PCFT by BioGEE in PCFT mutants. After transfection, cells were grown in medium containing 22 µM BioGEE in the presence or absence of 2 mM GSSG for 24 h. D : glutathionylation assays in a wider spectrum of PCFT mutants and PCFT-WT. For all panels, DTT-free sample buffer was used to process all samples and the images shown are representative of two independent analyses.
    Figure Legend Snippet: Glutathionylation of the substituted cysteine residues in PCFT-mutants. Panel A : Glutathionylation of PCFT mutants that were found to be posttranslationally modified. After transient transfection, cells were grown in RPMI-1640 medium containing 22 µM BioGEE for 24 h before cells were processed for pull-down assay with streptavidin beads. The last lane of this panel serves as a quantitative control in which PCFT expressed on the plasma membrane (P.M.) was labeled with EZ-Link Sulfo-NHS-LC-Biotin. B : the effects of DTT on the removal of the glutathionylation modification from PCFT mutants. Transient transfectants were labeled with BioGEE for 24 h and then treated with DTT (DTT+) or buffer alone (DTT−) before pull-down assays were performed with streptavidin beads. C : the effects of addition of GSSG on biotinylation of PCFT by BioGEE in PCFT mutants. After transfection, cells were grown in medium containing 22 µM BioGEE in the presence or absence of 2 mM GSSG for 24 h. D : glutathionylation assays in a wider spectrum of PCFT mutants and PCFT-WT. For all panels, DTT-free sample buffer was used to process all samples and the images shown are representative of two independent analyses.

    Techniques Used: Modification, Transfection, Pull Down Assay, Labeling

    27) Product Images from "T cell receptor-dependent S-acylation of ZAP-70 controls activation of T cells"

    Article Title: T cell receptor-dependent S-acylation of ZAP-70 controls activation of T cells

    Journal: bioRxiv

    doi: 10.1101/2020.06.30.180885

    ZAP-70 is S-acylated at cysteine 564. (A) Identification of S-acylation targets in Jurkat T cells. S-acylated proteins were captured using ABE and detected using protein-specific antibodies. Samples not treated with hydroxylamine (-HA) were used to estimate nonspecific binding with streptavidin resin. Lck and LAT were used as the positive control. PLC-γ1, GRB2, and ZAP-70 were identified as novel S-acylated proteins. Input samples shown were collected before addition of streptavidin. (B) Identification of ZAP-70 S-acylation site. ABE was performed on P116 cells stably expressing WT or mutant versions of ZAP-70. Loss of ZAP-70 S-acylation was observed in cells expressing ZAP-70 with C564R mutation and ZAP-70 with C560R, C564R double-mutation. S-acylation of GAPDH, a known S-acylated protein, was not affected and served as a positive and loading control. (C) Kinetics of agonist-induced S-acylation. Jurkat cells were stimulated with 10 µg anti-CD3 antibody for the indicated times and lysates were subject to ABE analysis. Calnexin, a known S-acylated protein was used as a positive and loading control. Input samples were used to confirm phosphorylation of T cell signaling proteins in response to T cell stimulation.
    Figure Legend Snippet: ZAP-70 is S-acylated at cysteine 564. (A) Identification of S-acylation targets in Jurkat T cells. S-acylated proteins were captured using ABE and detected using protein-specific antibodies. Samples not treated with hydroxylamine (-HA) were used to estimate nonspecific binding with streptavidin resin. Lck and LAT were used as the positive control. PLC-γ1, GRB2, and ZAP-70 were identified as novel S-acylated proteins. Input samples shown were collected before addition of streptavidin. (B) Identification of ZAP-70 S-acylation site. ABE was performed on P116 cells stably expressing WT or mutant versions of ZAP-70. Loss of ZAP-70 S-acylation was observed in cells expressing ZAP-70 with C564R mutation and ZAP-70 with C560R, C564R double-mutation. S-acylation of GAPDH, a known S-acylated protein, was not affected and served as a positive and loading control. (C) Kinetics of agonist-induced S-acylation. Jurkat cells were stimulated with 10 µg anti-CD3 antibody for the indicated times and lysates were subject to ABE analysis. Calnexin, a known S-acylated protein was used as a positive and loading control. Input samples were used to confirm phosphorylation of T cell signaling proteins in response to T cell stimulation.

    Techniques Used: Binding Assay, Positive Control, Planar Chromatography, Stable Transfection, Expressing, Mutagenesis, Cell Stimulation

    28) Product Images from "Heat Shock Protein 70 Inhibitors. 1. 2,5′-Thiodipyrimidine and 5-(Phenylthio)pyrimidine Acrylamides as Irreversible Binders to an Allosteric Site on Heat Shock Protein 70"

    Article Title: Heat Shock Protein 70 Inhibitors. 1. 2,5′-Thiodipyrimidine and 5-(Phenylthio)pyrimidine Acrylamides as Irreversible Binders to an Allosteric Site on Heat Shock Protein 70

    Journal: Journal of Medicinal Chemistry

    doi: 10.1021/jm401551n

    Designed ligands interact specifically with Hsp70 in cancer cells. (a) Streptavidin beads were incubated with the indicated concentrations of 44b , 45b , and d -biotin, the unbound agent was washed off, and the resulting beads carrying 44b , 45b , or d -biotin were probed with SKBr3 cell extracts (500 μg). Hsp70 isolated on the beads was probed by Western blot (WB). A representative blot is presented (top). Blots were quantified by densitometry and values, in relative luminescence units, graphed against the concentration of added biotinylated Hsp70 inhibitor (bottom). Results from three independent experiments were graphed to determine the relative binding affinity ( K d ) of 44b and 45b using equations as implemented in the GraphPad Prism software. Key: points, mean; bars, SD. (b) Cells were treated with the indicated concentrations of 44b for 6 h prior to lysing and precipitation of protein complexes on streptavidin beads. Beads were washed with high-salt (1 M NaCl) buffer before elution of proteins on a denaturing gel and silver staining. BB70 is an antibody specific for Hsp70. This antibody also recognizes Grp78 and Grp75, the endoplasmic reticulum and the mitochondrial Hsp70 paralogues, respectively. (c) Protein complexes were isolated as indicated in (b) in cells pretreated with 17a . CP = chemical precipitation. (d) Analysis of the Hsp70–HOP complex. SKBr3 cells were treated for 24 h with vehicle or indicated concentrations of 20a . Upon cell lysing, Hsp70 complexes were isolated with an anti-Hsp70 antibody (IP BB70) and analyzed by WB. Specificity of binding was tested with a control IgG. Gels were quantified by densitometry, values normalized to the control (vehicle only treated cells), and data graphed against the concentration of 20a . Error bars represent the SEM ( n = 2).
    Figure Legend Snippet: Designed ligands interact specifically with Hsp70 in cancer cells. (a) Streptavidin beads were incubated with the indicated concentrations of 44b , 45b , and d -biotin, the unbound agent was washed off, and the resulting beads carrying 44b , 45b , or d -biotin were probed with SKBr3 cell extracts (500 μg). Hsp70 isolated on the beads was probed by Western blot (WB). A representative blot is presented (top). Blots were quantified by densitometry and values, in relative luminescence units, graphed against the concentration of added biotinylated Hsp70 inhibitor (bottom). Results from three independent experiments were graphed to determine the relative binding affinity ( K d ) of 44b and 45b using equations as implemented in the GraphPad Prism software. Key: points, mean; bars, SD. (b) Cells were treated with the indicated concentrations of 44b for 6 h prior to lysing and precipitation of protein complexes on streptavidin beads. Beads were washed with high-salt (1 M NaCl) buffer before elution of proteins on a denaturing gel and silver staining. BB70 is an antibody specific for Hsp70. This antibody also recognizes Grp78 and Grp75, the endoplasmic reticulum and the mitochondrial Hsp70 paralogues, respectively. (c) Protein complexes were isolated as indicated in (b) in cells pretreated with 17a . CP = chemical precipitation. (d) Analysis of the Hsp70–HOP complex. SKBr3 cells were treated for 24 h with vehicle or indicated concentrations of 20a . Upon cell lysing, Hsp70 complexes were isolated with an anti-Hsp70 antibody (IP BB70) and analyzed by WB. Specificity of binding was tested with a control IgG. Gels were quantified by densitometry, values normalized to the control (vehicle only treated cells), and data graphed against the concentration of 20a . Error bars represent the SEM ( n = 2).

    Techniques Used: Incubation, Isolation, Western Blot, Concentration Assay, Binding Assay, Software, Silver Staining

    29) Product Images from "Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium"

    Article Title: Acute inhibition of centriolar satellite function and positioning reveals their functions at the primary cilium

    Journal: bioRxiv

    doi: 10.1101/2020.02.10.941658

    The proximity interactome of PCM1 at the peripheral clusters is enriched for proteins implicated in ciliogenesis and mitosis The proximity PCM1 interactome of satellites were identified using the BioID approach. (A) HEK293T cells were transfected with Myc-BirA*-PCM1-FKBP and induced for peripheral targeting of satellites with rapamycin treatment for 1 h. Cells that were not treated with rapamycin were used as a control. After 18 h biotin incubation, cells were fixed and stained for Myc-BirA*-PCM1-FKBP expression with anti-myc, biotinylated proteins with streptavidin and centrosomes with g-tubulin. DNA was stained with DAPI. Scale bar = 10 μm, cell edges are outlined. (B) Biotinylated proteins from cell lysates from cells expressing Myc-BirA*-PCM1-FKBP (-rapamycin or +rapamycin) were pulled down with streptavidin chromatography and samples were analyzed by SDS-PAGE and western blotting with streptavidin to detect biotinylated proteins and with anti-Cep131 (positive control). IS: initial sample used for streptavidin pulldowns, Beads: Eluted proteins. (C, D) GO-enrichment analysis of the proximity interactors of PCM1 after rapamycin treatment based on their (C) biological processes and (D) cellular compartment. The X-axis represents the log transformed p-value (Fishers exact test) of GO terms. (E) The cilium-associated proteins in the interactome of peripheral satellites were determined based on GO categories and previous work and different ciliogenesis functional modules plotted in the “peripheral PCM1 interaction network using Cytoscape.
    Figure Legend Snippet: The proximity interactome of PCM1 at the peripheral clusters is enriched for proteins implicated in ciliogenesis and mitosis The proximity PCM1 interactome of satellites were identified using the BioID approach. (A) HEK293T cells were transfected with Myc-BirA*-PCM1-FKBP and induced for peripheral targeting of satellites with rapamycin treatment for 1 h. Cells that were not treated with rapamycin were used as a control. After 18 h biotin incubation, cells were fixed and stained for Myc-BirA*-PCM1-FKBP expression with anti-myc, biotinylated proteins with streptavidin and centrosomes with g-tubulin. DNA was stained with DAPI. Scale bar = 10 μm, cell edges are outlined. (B) Biotinylated proteins from cell lysates from cells expressing Myc-BirA*-PCM1-FKBP (-rapamycin or +rapamycin) were pulled down with streptavidin chromatography and samples were analyzed by SDS-PAGE and western blotting with streptavidin to detect biotinylated proteins and with anti-Cep131 (positive control). IS: initial sample used for streptavidin pulldowns, Beads: Eluted proteins. (C, D) GO-enrichment analysis of the proximity interactors of PCM1 after rapamycin treatment based on their (C) biological processes and (D) cellular compartment. The X-axis represents the log transformed p-value (Fishers exact test) of GO terms. (E) The cilium-associated proteins in the interactome of peripheral satellites were determined based on GO categories and previous work and different ciliogenesis functional modules plotted in the “peripheral PCM1 interaction network using Cytoscape.

    Techniques Used: Transfection, Incubation, Staining, Expressing, Chromatography, SDS Page, Western Blot, Positive Control, Transformation Assay, Functional Assay

    30) Product Images from "LOTUS domain is a novel class of G-rich and G-quadruplex RNA binding domain"

    Article Title: LOTUS domain is a novel class of G-rich and G-quadruplex RNA binding domain

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa652

    TDRD5 LOTUS domains bind to G-rich RNA. ( A ) Domain architecture of mammalian TDRD5. Three LOTUS domains are abbreviated as L1, L2 and L3, respectively. ( B ) Purification of mouse TDRD5 LOTUS domains. His-tagged LOTUS domains were purified by affinity chromatography followed by gel filtration chromatography. ( C ) LOTUS domains specifically bind to poly(G) RNA. Biotin-labeled oligonucleotides were bound to streptavidin beads. After incubating with purified His-tagged LOTUS domains, Western blotting using an anti-His antibody was performed to detect bound LOTUS domains. Biotin-labeled oligonucleotide inputs were measured by dot blot analysis. ( D ) LOTUS domains specifically bind to poly(G) RNA by ELISA assays. n = 3, error bars represent s.e.m. Dissociation constants ( K d ) are indicated. ( E ) poly(G) RNA binds endogenous TDRD5 from mouse testes. Biotin-labeled poly(G) RNA oligonucleotides were bound to streptavidin beads. After incubating with adult mouse testis lysates, Western blotting was performed using TDRD5 and β-actin antibodies. TDRD5 isoform1 and isoform2 were shown. Asterisk shows a non-specific band. ( F ) LOTUS domains bind to G-rich RNA. Biotin-labeled oligonucleotides were bound to streptavidin beads. After incubating with purified His-tagged LOTUS domains, Western blotting using an anti-His antibody was performed to detect bound LOTUS domains. Biotin-labeled oligonucleotide inputs were measured by dot blot analysis. ( G ) LOTUS domains but not the Tudor domain of TDRD5 bind to G-rich RNA. HEK293T cells were transfected with plasmids coding for GFP-tagged full-length (FL) or truncated (N or C) TDRD5. Biotin-labeled oligonucleotides were bound to streptavidin beads. After incubating with HEK293T cell lysates, Western blotting was performed using an anti-GFP antibody.
    Figure Legend Snippet: TDRD5 LOTUS domains bind to G-rich RNA. ( A ) Domain architecture of mammalian TDRD5. Three LOTUS domains are abbreviated as L1, L2 and L3, respectively. ( B ) Purification of mouse TDRD5 LOTUS domains. His-tagged LOTUS domains were purified by affinity chromatography followed by gel filtration chromatography. ( C ) LOTUS domains specifically bind to poly(G) RNA. Biotin-labeled oligonucleotides were bound to streptavidin beads. After incubating with purified His-tagged LOTUS domains, Western blotting using an anti-His antibody was performed to detect bound LOTUS domains. Biotin-labeled oligonucleotide inputs were measured by dot blot analysis. ( D ) LOTUS domains specifically bind to poly(G) RNA by ELISA assays. n = 3, error bars represent s.e.m. Dissociation constants ( K d ) are indicated. ( E ) poly(G) RNA binds endogenous TDRD5 from mouse testes. Biotin-labeled poly(G) RNA oligonucleotides were bound to streptavidin beads. After incubating with adult mouse testis lysates, Western blotting was performed using TDRD5 and β-actin antibodies. TDRD5 isoform1 and isoform2 were shown. Asterisk shows a non-specific band. ( F ) LOTUS domains bind to G-rich RNA. Biotin-labeled oligonucleotides were bound to streptavidin beads. After incubating with purified His-tagged LOTUS domains, Western blotting using an anti-His antibody was performed to detect bound LOTUS domains. Biotin-labeled oligonucleotide inputs were measured by dot blot analysis. ( G ) LOTUS domains but not the Tudor domain of TDRD5 bind to G-rich RNA. HEK293T cells were transfected with plasmids coding for GFP-tagged full-length (FL) or truncated (N or C) TDRD5. Biotin-labeled oligonucleotides were bound to streptavidin beads. After incubating with HEK293T cell lysates, Western blotting was performed using an anti-GFP antibody.

    Techniques Used: Purification, Affinity Chromatography, Filtration, Chromatography, Labeling, Western Blot, Dot Blot, Enzyme-linked Immunosorbent Assay, Transfection

    TDRD5 LOTUS domains bind to folded RNA G-quadruplex structure. ( A ) Sequences of different RNA G4s. Schematic representation of a RNA G-quadruplex (G4) formed by a G-rich sequence was shown at the top. ( B ) In vitro RNA pull-down analysis of TDRD5 LOTUS domains. Biotin-labeled RNA G4 oligonucleotides and their respective mutants (mut) were used in the RNA pull-down assay as described in (A). ( C ) TDRD5 LOTUS domains bind RNA G4 with high affinity. ELISA assay was performed with three individual TDRD5 LOTUS domains (L1, L2 and L3) and indicated RNA oligos. The concentration (conc.) of LOTUS domains used is shown on the x-axis of each graph. TDRD5 L1 and L2 but not L3 bind RNA G4. n = 3, error bars represent s.e.m. Dissociation constants ( K d ) are indicated. ( D ) Circular dichroism spectroscopy of TERRA G4 RNA oligos in KCl or LiCl buffers. ( E ) ELISA assay was performed with TDRD5 LOTUS domains and TERRA G4 RNA oligos in KCl or LiCl buffers. n = 3, error bars represent s.e.m. Dissociation constants ( K d ) are indicated. ( F ) RNA G4 binds ectopically expressed full-length TDRD5. HEK293T cells were transfected with GFP-TDRD5 plasmid or GFP empty plasmid as a negative control. Biotin-labeled RNA G4 oligonucleotides were annealed and bound to streptavidin beads. After incubating with cell lysates, Western blotting was performed with an anti-GFP antibody. ( G ) RNA G4 binds endogenous TDRD5 from mouse testes. Biotin-labeled G4 oligonucleotides were annealed and bound to streptavidin beads. After incubating with adult mouse testis lysates, Western blotting was performed with TDRD5 and β-actin antibodies. TDRD5 isoform1 and isoform2 are shown.
    Figure Legend Snippet: TDRD5 LOTUS domains bind to folded RNA G-quadruplex structure. ( A ) Sequences of different RNA G4s. Schematic representation of a RNA G-quadruplex (G4) formed by a G-rich sequence was shown at the top. ( B ) In vitro RNA pull-down analysis of TDRD5 LOTUS domains. Biotin-labeled RNA G4 oligonucleotides and their respective mutants (mut) were used in the RNA pull-down assay as described in (A). ( C ) TDRD5 LOTUS domains bind RNA G4 with high affinity. ELISA assay was performed with three individual TDRD5 LOTUS domains (L1, L2 and L3) and indicated RNA oligos. The concentration (conc.) of LOTUS domains used is shown on the x-axis of each graph. TDRD5 L1 and L2 but not L3 bind RNA G4. n = 3, error bars represent s.e.m. Dissociation constants ( K d ) are indicated. ( D ) Circular dichroism spectroscopy of TERRA G4 RNA oligos in KCl or LiCl buffers. ( E ) ELISA assay was performed with TDRD5 LOTUS domains and TERRA G4 RNA oligos in KCl or LiCl buffers. n = 3, error bars represent s.e.m. Dissociation constants ( K d ) are indicated. ( F ) RNA G4 binds ectopically expressed full-length TDRD5. HEK293T cells were transfected with GFP-TDRD5 plasmid or GFP empty plasmid as a negative control. Biotin-labeled RNA G4 oligonucleotides were annealed and bound to streptavidin beads. After incubating with cell lysates, Western blotting was performed with an anti-GFP antibody. ( G ) RNA G4 binds endogenous TDRD5 from mouse testes. Biotin-labeled G4 oligonucleotides were annealed and bound to streptavidin beads. After incubating with adult mouse testis lysates, Western blotting was performed with TDRD5 and β-actin antibodies. TDRD5 isoform1 and isoform2 are shown.

    Techniques Used: Sequencing, In Vitro, Labeling, Pull Down Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Spectroscopy, Transfection, Plasmid Preparation, Negative Control, Western Blot

    31) Product Images from "Lysine 48-Linked Polyubiquitination of Organic Anion Transporter-1 Is Essential for Its Protein Kinase C-Regulated Endocytosis"

    Article Title: Lysine 48-Linked Polyubiquitination of Organic Anion Transporter-1 Is Essential for Its Protein Kinase C-Regulated Endocytosis

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.112.082065

    Effect of ubiquitin mutant Ub-K48R on PMA-regulated OAT1 surface expression. (A) OAT1-expressing cells were transfected with Ub-K48R (or vector as control), followed by treatment with or without PMA (1 μ M) for 30 minutes. The treated cells underwent cell-surface biotinylation. Biotinylated proteins were isolated with streptavidin beads and analyzed by immunoblotting with an anti-myc antibody. (B) densitometry plot of results from (A) as well as from other experiments. Surface OAT1 in PMA-treated cells was expressed as the percentage of surface OAT1 in control cells. The values are mean ± S.E. ( n = 3).
    Figure Legend Snippet: Effect of ubiquitin mutant Ub-K48R on PMA-regulated OAT1 surface expression. (A) OAT1-expressing cells were transfected with Ub-K48R (or vector as control), followed by treatment with or without PMA (1 μ M) for 30 minutes. The treated cells underwent cell-surface biotinylation. Biotinylated proteins were isolated with streptavidin beads and analyzed by immunoblotting with an anti-myc antibody. (B) densitometry plot of results from (A) as well as from other experiments. Surface OAT1 in PMA-treated cells was expressed as the percentage of surface OAT1 in control cells. The values are mean ± S.E. ( n = 3).

    Techniques Used: Mutagenesis, Expressing, Transfection, Plasmid Preparation, Isolation

    32) Product Images from "Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting"

    Article Title: Detergent-insoluble GPI-anchored Proteins Are Apically Sorted in Fischer Rat Thyroid Cells, but Interference with Cholesterol or Sphingolipids Differentially Affects Detergent Insolubility and Apical Sorting

    Journal: Molecular Biology of the Cell

    doi:

    Extraction in TX-100 of apical and basolateral PLAP in transfected FRT cells. (A) FRT cells expressing PLAP were labeled with [ 35 S]met–cys for 30 min and chased for 90 or 120 min in chase medium. Cells were then biotinylated from the apical (Ap) and basolateral (BL) domains, lysed in TNE/1% TX-100 buffer at 4°C, and separated by centrifugation into soluble (S; supernatant) and insoluble (I; pellet) fractions that were immunoprecipitated with a specific antibody against PLAP and reprecipitated with streptavidin. Insoluble PLAP is present on the apical and basolateral surfaces at similar rates. (B) Quantification of three independent experiments is shown as mean ± SD. Hatched bars represent soluble proteins, and black bars represent insoluble proteins.
    Figure Legend Snippet: Extraction in TX-100 of apical and basolateral PLAP in transfected FRT cells. (A) FRT cells expressing PLAP were labeled with [ 35 S]met–cys for 30 min and chased for 90 or 120 min in chase medium. Cells were then biotinylated from the apical (Ap) and basolateral (BL) domains, lysed in TNE/1% TX-100 buffer at 4°C, and separated by centrifugation into soluble (S; supernatant) and insoluble (I; pellet) fractions that were immunoprecipitated with a specific antibody against PLAP and reprecipitated with streptavidin. Insoluble PLAP is present on the apical and basolateral surfaces at similar rates. (B) Quantification of three independent experiments is shown as mean ± SD. Hatched bars represent soluble proteins, and black bars represent insoluble proteins.

    Techniques Used: Transfection, Expressing, Labeling, Centrifugation, Immunoprecipitation

    Surface expression of GPI-anchored, secretory, and transmembrane proteins after FB1 treatment. (A) FRT cells stably expressing PLAP, NTR-PLAP, or gD1-DAF were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After 30 min of labeling with [ 35 S]met–cys or [ 35 S]cys and a 2-h chase, cells were surface biotinylated from the apical (Ap) or the basolateral (BL) domain, lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (B) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. Apical PLAP and NTR-PLAP, but not basolateral gD1-DAF, became unpolarized after FB1 treatment. (C) FRT cells expressing PLAP-sec, NTR-sec, p75NTR, or PLAP-PS321 were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After labeling with [ 35 S]met–cys or [ 35 S]cys for 20 min and a chase of 2 h, apical and basolateral media were collected separately and cells were surface biotinylated from apical or basolateral domains. Polarized secretion of the secretory forms was revealed by immunoprecipitation in the apical and basolateral medium (top two panels), whereas biotinylated transmembrane proteins were revealed by double immunoprecipitation with specific antibody and streptavidin beads (bottom two panels). (D) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. FB1 does not alter the sorting of transmembrane and secretory proteins.
    Figure Legend Snippet: Surface expression of GPI-anchored, secretory, and transmembrane proteins after FB1 treatment. (A) FRT cells stably expressing PLAP, NTR-PLAP, or gD1-DAF were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After 30 min of labeling with [ 35 S]met–cys or [ 35 S]cys and a 2-h chase, cells were surface biotinylated from the apical (Ap) or the basolateral (BL) domain, lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (B) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. Apical PLAP and NTR-PLAP, but not basolateral gD1-DAF, became unpolarized after FB1 treatment. (C) FRT cells expressing PLAP-sec, NTR-sec, p75NTR, or PLAP-PS321 were grown to confluence in the absence (−) or presence (+) of FB1, as described in MATERIALS AND METHODS. After labeling with [ 35 S]met–cys or [ 35 S]cys for 20 min and a chase of 2 h, apical and basolateral media were collected separately and cells were surface biotinylated from apical or basolateral domains. Polarized secretion of the secretory forms was revealed by immunoprecipitation in the apical and basolateral medium (top two panels), whereas biotinylated transmembrane proteins were revealed by double immunoprecipitation with specific antibody and streptavidin beads (bottom two panels). (D) Amounts of labeled proteins were quantified from three independent experiments and are shown as means ± SD. Hatched bars represent apical proteins, and black bars represent basolateral proteins. FB1 does not alter the sorting of transmembrane and secretory proteins.

    Techniques Used: Expressing, Stable Transfection, Labeling, Immunoprecipitation, Size-exclusion Chromatography

    Steady-state distribution and delivery to the plasma membrane of PLAP and NTR-PLAP in transfected FRT cells. (A) Schemes of PLAP and NTR-PLAP (a fusion protein containing the ectodomain of p75NTR and the C-terminal GPI attachment signal of PLAP). (B) FRT cells stably expressing PLAP or NTR-PLAP were grown to confluence and pulsed overnight with [ 35 S]met–cys or [ 35 S]cys, respectively. After surface biotinylation from the apical (Ap) and basolateral (BL) domains, cells were lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (C) FRT cells were labeled for 20 min with [ 35 S]met–cys or [ 35 S]cys. At different times of the chase, surface proteins were biotinylated from the apical (Ap) or the basolateral (BL) side. Cells were then lysed and immunoprecipitated with antibodies against PLAP or NTR and with streptavidin beads. Samples were run on 10% SDS-PAGE and detected by fluorography. PLAP and NTR-PLAP are directly sorted to the apical surface in FRT cells, whereas a small percentage of the two proteins is delivered with similar kinetics to the basolateral domain.
    Figure Legend Snippet: Steady-state distribution and delivery to the plasma membrane of PLAP and NTR-PLAP in transfected FRT cells. (A) Schemes of PLAP and NTR-PLAP (a fusion protein containing the ectodomain of p75NTR and the C-terminal GPI attachment signal of PLAP). (B) FRT cells stably expressing PLAP or NTR-PLAP were grown to confluence and pulsed overnight with [ 35 S]met–cys or [ 35 S]cys, respectively. After surface biotinylation from the apical (Ap) and basolateral (BL) domains, cells were lysed, immunoprecipitated against specific antibodies, and reprecipitated against streptavidin. (C) FRT cells were labeled for 20 min with [ 35 S]met–cys or [ 35 S]cys. At different times of the chase, surface proteins were biotinylated from the apical (Ap) or the basolateral (BL) side. Cells were then lysed and immunoprecipitated with antibodies against PLAP or NTR and with streptavidin beads. Samples were run on 10% SDS-PAGE and detected by fluorography. PLAP and NTR-PLAP are directly sorted to the apical surface in FRT cells, whereas a small percentage of the two proteins is delivered with similar kinetics to the basolateral domain.

    Techniques Used: Transfection, Stable Transfection, Expressing, Immunoprecipitation, Labeling, SDS Page

    33) Product Images from "Is Phosphorylation of the ?1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester-activated Protein Kinase C?"

    Article Title: Is Phosphorylation of the ?1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester-activated Protein Kinase C?

    Journal: Molecular Biology of the Cell

    doi:

    Incubation at 18°C prevents the PDBu-induced down-regulation of cell surface Na,K-ATPase. COS-7 cells expressing wild-type Bufo α1 subunit were preincubated for 30 min at 18°C in the absence or in the presence of 10 −7 M PDBu (P), 5 × 10 −7 M GF109203X (G), or PDBu and GF109203X (G+P) before biotinylation of cell surface proteins (A and B) or measurement of exogenous Na,K-ATPase-mediated 86 Rb uptake (C). After streptavidin precipitation, the cell surface–expressed Bufo α1 subunit was detected by immunoblot using a specific anti- Bufo α1 subunit antibody. (A) A representative immunoblot is shown (n = 4). (B) Quantitation of the relative amounts of Na,K-ATPase α1 subunit detected by immunoblotting. Results are expressed as fractional change (with respect to control value) and are means ± SE from four independent experiments. (C) Ouabain-sensitive 86 Rb uptake. Results are expressed as fractional change (with respect to control value) and are means ± SE from 11 independent experiments (**, p
    Figure Legend Snippet: Incubation at 18°C prevents the PDBu-induced down-regulation of cell surface Na,K-ATPase. COS-7 cells expressing wild-type Bufo α1 subunit were preincubated for 30 min at 18°C in the absence or in the presence of 10 −7 M PDBu (P), 5 × 10 −7 M GF109203X (G), or PDBu and GF109203X (G+P) before biotinylation of cell surface proteins (A and B) or measurement of exogenous Na,K-ATPase-mediated 86 Rb uptake (C). After streptavidin precipitation, the cell surface–expressed Bufo α1 subunit was detected by immunoblot using a specific anti- Bufo α1 subunit antibody. (A) A representative immunoblot is shown (n = 4). (B) Quantitation of the relative amounts of Na,K-ATPase α1 subunit detected by immunoblotting. Results are expressed as fractional change (with respect to control value) and are means ± SE from four independent experiments. (C) Ouabain-sensitive 86 Rb uptake. Results are expressed as fractional change (with respect to control value) and are means ± SE from 11 independent experiments (**, p

    Techniques Used: Incubation, Expressing, Quantitation Assay

    34) Product Images from "Citrullination of NF-kB p65 enhances its nuclear localization and TLR-induced expression of IL-1β and TNFα"

    Article Title: Citrullination of NF-kB p65 enhances its nuclear localization and TLR-induced expression of IL-1β and TNFα

    Journal: Science immunology

    doi: 10.1126/sciimmunol.aal3062

    Citrullination of p65 in vivo and in vitro A B . Whole cell extract from dHL60 cells stimulated with LPS in the presence or absence of BB-Cl-amidine (BB-Cl) pre-treatment was labeled with biotin-PG probe, pulled down with streptavidin beads, and probed with anti-histone H3 (H3) ( A ) or anti-p65 ( B ) in western blotting (the top panels). A fraction of the biotin-PG-labeled extract prior to pull-down was also analyzed to serve as input controls. The normalized density of pulldown H3 or p65 from two experiments is shown in the dot graph. Data from the same experiment are connected with lines. C–E . Recombinant GST, GST-p65, and various R-to-K mutants were incubated with ( C–E ) or without ( C ) PAD4, fractionated by SDS-PAGE gels, stained with Coomassie blue (the left panels) or probed with F95 (the right panels of C and E , and the middle panel of D ) in western blotting. Schematic diagrams of predicted recombinant GST-p65 proteins are shown in the right panel of D . Arginine residues are marked with *. The diagrams and the location of the arginine residues are not to scale. F . The density of the citrullinated 50–80 kD bands detected with F95 was normalized against that of the corresponding bands detected with Coomassie blue. The normalized density of citrullinated WT GST-p65 was arbitrarily set as 1. Cumulated results from all experiments are shown. Each dot represents one experiment.
    Figure Legend Snippet: Citrullination of p65 in vivo and in vitro A B . Whole cell extract from dHL60 cells stimulated with LPS in the presence or absence of BB-Cl-amidine (BB-Cl) pre-treatment was labeled with biotin-PG probe, pulled down with streptavidin beads, and probed with anti-histone H3 (H3) ( A ) or anti-p65 ( B ) in western blotting (the top panels). A fraction of the biotin-PG-labeled extract prior to pull-down was also analyzed to serve as input controls. The normalized density of pulldown H3 or p65 from two experiments is shown in the dot graph. Data from the same experiment are connected with lines. C–E . Recombinant GST, GST-p65, and various R-to-K mutants were incubated with ( C–E ) or without ( C ) PAD4, fractionated by SDS-PAGE gels, stained with Coomassie blue (the left panels) or probed with F95 (the right panels of C and E , and the middle panel of D ) in western blotting. Schematic diagrams of predicted recombinant GST-p65 proteins are shown in the right panel of D . Arginine residues are marked with *. The diagrams and the location of the arginine residues are not to scale. F . The density of the citrullinated 50–80 kD bands detected with F95 was normalized against that of the corresponding bands detected with Coomassie blue. The normalized density of citrullinated WT GST-p65 was arbitrarily set as 1. Cumulated results from all experiments are shown. Each dot represents one experiment.

    Techniques Used: In Vivo, In Vitro, Labeling, Western Blot, Recombinant, Incubation, SDS Page, Staining

    35) Product Images from "Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer"

    Article Title: Long noncoding RNA AFAP1-AS1 facilitates tumor growth through enhancer of zeste homolog 2 in colorectal cancer

    Journal: American Journal of Cancer Research

    doi:

    Association of AFAP1-AS1 and Polycomb Repressive Complex 2. (A) RIP enrichment was determined as RNA associated with EZH2 IP relative to an input control. (B) RIP experiments were performed using the EZH2 antibody to immunoprecipitate (IP). (C) Biotinylated AFAP1-AS1 was incubated with nuclear extracts (SW480 and HCT116 cells), targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and AFAP1-AS1 (n = 3). (D, E) RNAs corresponding to different fragments of AFAP1-AS1 were treated as in (C), and associated EZH2 was detected by western blotting (n = 3). Error bars ± SD. *, P
    Figure Legend Snippet: Association of AFAP1-AS1 and Polycomb Repressive Complex 2. (A) RIP enrichment was determined as RNA associated with EZH2 IP relative to an input control. (B) RIP experiments were performed using the EZH2 antibody to immunoprecipitate (IP). (C) Biotinylated AFAP1-AS1 was incubated with nuclear extracts (SW480 and HCT116 cells), targeted with streptavidin beads, and washed, and associated proteins were resolved in a gel. Western blotting analysis of the specific association of EZH2 and AFAP1-AS1 (n = 3). (D, E) RNAs corresponding to different fragments of AFAP1-AS1 were treated as in (C), and associated EZH2 was detected by western blotting (n = 3). Error bars ± SD. *, P

    Techniques Used: Incubation, Western Blot

    36) Product Images from "Identification of Amino Acids Essential for Estrone-3-Sulfate Transport within Transmembrane Domain 2 of Organic Anion Transporting Polypeptide 1B1"

    Article Title: Identification of Amino Acids Essential for Estrone-3-Sulfate Transport within Transmembrane Domain 2 of Organic Anion Transporting Polypeptide 1B1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0036647

    Protein expression of mutants with reduced transport activity. A. Cell surface expression of OATP1B1 and mutants. Upper panel, representative blot of OATP1B1 and mutants (D70A, F73A, E74A, G76A). Lower panel, the intensity was quantified relative to the wild-type. Cells were biotinylated, and the biotin-labeled cell surface proteins were precipitated with streptavidin beads, separated by SDS-PAGE, followed by Western blotting with anti-HA antibody. Same blot was probed with integrin antibody as surface protein loading control. B. Total protein expression of OATP1B1 and mutants. Cells were lysed with RIPA buffer, separated by SDS-PAGE, followed by Western blotting with anti-HA antibody. The band intensity was quantified relative to the wild-type. The results shown are means ± S.E. ( n = 3).
    Figure Legend Snippet: Protein expression of mutants with reduced transport activity. A. Cell surface expression of OATP1B1 and mutants. Upper panel, representative blot of OATP1B1 and mutants (D70A, F73A, E74A, G76A). Lower panel, the intensity was quantified relative to the wild-type. Cells were biotinylated, and the biotin-labeled cell surface proteins were precipitated with streptavidin beads, separated by SDS-PAGE, followed by Western blotting with anti-HA antibody. Same blot was probed with integrin antibody as surface protein loading control. B. Total protein expression of OATP1B1 and mutants. Cells were lysed with RIPA buffer, separated by SDS-PAGE, followed by Western blotting with anti-HA antibody. The band intensity was quantified relative to the wild-type. The results shown are means ± S.E. ( n = 3).

    Techniques Used: Expressing, Activity Assay, Labeling, SDS Page, Western Blot

    37) Product Images from "Post-Transcriptional Regulation of Alpha One Antitrypsin by a Proteasome Inhibitor"

    Article Title: Post-Transcriptional Regulation of Alpha One Antitrypsin by a Proteasome Inhibitor

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21124318

    MG132 causes stress granule formation and promotes RNA binding proteins associated with α1AT mRNA. ( A ) C3A cells were plated on type I collagen-coated glass coverslips and treated with MG132 for 2 h. The cells were stained with G3BP1 antibody and mounted using the Prolong Gold Antifade reagent with DAPI. The positive G3BP1 puncta which represent stress granule were denoted by an arrow (Scale bar = 10 μm). ( B ) Biotinylated α1AT RNA incubated in 500 µg C3A cytoplasmic protein (Ctrl and MG132 treatment). The RNA-RBPs complex were pulled down with streptavidin agarose beads and applied for western blot analysis. ( C ) C3A cells were transiently transfected with empty vector or GFP tagged G3BP1 for 48 h and then treated with MG132. Cell lysates were analyzed by western blot analysis using indicated antibodies.
    Figure Legend Snippet: MG132 causes stress granule formation and promotes RNA binding proteins associated with α1AT mRNA. ( A ) C3A cells were plated on type I collagen-coated glass coverslips and treated with MG132 for 2 h. The cells were stained with G3BP1 antibody and mounted using the Prolong Gold Antifade reagent with DAPI. The positive G3BP1 puncta which represent stress granule were denoted by an arrow (Scale bar = 10 μm). ( B ) Biotinylated α1AT RNA incubated in 500 µg C3A cytoplasmic protein (Ctrl and MG132 treatment). The RNA-RBPs complex were pulled down with streptavidin agarose beads and applied for western blot analysis. ( C ) C3A cells were transiently transfected with empty vector or GFP tagged G3BP1 for 48 h and then treated with MG132. Cell lysates were analyzed by western blot analysis using indicated antibodies.

    Techniques Used: RNA Binding Assay, Staining, Incubation, Western Blot, Transfection, Plasmid Preparation

    Related Articles

    Injection:

    Article Title: Progress of endocytic CHRN to autophagic degradation is regulated by RAB5-GTPase and T145 phosphorylation of SH3GLB1 at mouse neuromuscular junctions in vivo
    Article Snippet: Lysate dots were applied on PVDF membrane followed by immunodetection. .. For affinity coprecipitation ( ) gastrocnemius muscles were injected with 125 pmoles of BGT coupled with biotin (Invitrogen, B1196) and then harvested after 5 h. Tissue lysis was performed as mentioned above, while affinity precipitation with NeutrAvidin beads (Thermo Fisher Scientific, 29202) and western blot were performed as previously described. .. The following antibodies were used for the detection of their respective proteins: mouse anti-SH3GLB1 (Imgenex, IMG-265A), guinea pig anti-phospho-SH3GLB1 custom made with synthetic peptide (NFLpTPLRNFIC, PSL), rabbit anti-RAB5 (Cell Signalling Technology, 3547), mouse anti-GAPDH/glyceraldehyde-3-phosphate dehydrogenase (Thermo Fisher Scientific, MA5-15738), rabbit anti-ADRB2/adrenoceptor β 2 (Santa Cruz Biotechnology, sc-569), mouse anti-CHRN (BD Bioscience, 610989), rabbit anti-MAP1LC3B (Genetex, GTX127375), Living Colors® A.v. monoclonal antibody JL-8 (Clontech Laboratories, 632380), anti-mCherry (Acris, AP32117 PU), anti-USP6NL (Bethyl Laboratories, A302-794A-M), anti-RABGEF1 (Cell Signalling Technology, 7622), anti-rabbit coupled to horseradish peroxidase (HRP; Dako, P0448), anti-mouse-HRP (Thermo Fisher Scientific, 32430), anti-guinea pig-HRP (Thermo Fisher Scientific, PA1-28597), anti goat-HRP (Dako, P0449).

    Lysis:

    Article Title: Progress of endocytic CHRN to autophagic degradation is regulated by RAB5-GTPase and T145 phosphorylation of SH3GLB1 at mouse neuromuscular junctions in vivo
    Article Snippet: Lysate dots were applied on PVDF membrane followed by immunodetection. .. For affinity coprecipitation ( ) gastrocnemius muscles were injected with 125 pmoles of BGT coupled with biotin (Invitrogen, B1196) and then harvested after 5 h. Tissue lysis was performed as mentioned above, while affinity precipitation with NeutrAvidin beads (Thermo Fisher Scientific, 29202) and western blot were performed as previously described. .. The following antibodies were used for the detection of their respective proteins: mouse anti-SH3GLB1 (Imgenex, IMG-265A), guinea pig anti-phospho-SH3GLB1 custom made with synthetic peptide (NFLpTPLRNFIC, PSL), rabbit anti-RAB5 (Cell Signalling Technology, 3547), mouse anti-GAPDH/glyceraldehyde-3-phosphate dehydrogenase (Thermo Fisher Scientific, MA5-15738), rabbit anti-ADRB2/adrenoceptor β 2 (Santa Cruz Biotechnology, sc-569), mouse anti-CHRN (BD Bioscience, 610989), rabbit anti-MAP1LC3B (Genetex, GTX127375), Living Colors® A.v. monoclonal antibody JL-8 (Clontech Laboratories, 632380), anti-mCherry (Acris, AP32117 PU), anti-USP6NL (Bethyl Laboratories, A302-794A-M), anti-RABGEF1 (Cell Signalling Technology, 7622), anti-rabbit coupled to horseradish peroxidase (HRP; Dako, P0448), anti-mouse-HRP (Thermo Fisher Scientific, 32430), anti-guinea pig-HRP (Thermo Fisher Scientific, PA1-28597), anti goat-HRP (Dako, P0449).

    Affinity Precipitation:

    Article Title: Progress of endocytic CHRN to autophagic degradation is regulated by RAB5-GTPase and T145 phosphorylation of SH3GLB1 at mouse neuromuscular junctions in vivo
    Article Snippet: Lysate dots were applied on PVDF membrane followed by immunodetection. .. For affinity coprecipitation ( ) gastrocnemius muscles were injected with 125 pmoles of BGT coupled with biotin (Invitrogen, B1196) and then harvested after 5 h. Tissue lysis was performed as mentioned above, while affinity precipitation with NeutrAvidin beads (Thermo Fisher Scientific, 29202) and western blot were performed as previously described. .. The following antibodies were used for the detection of their respective proteins: mouse anti-SH3GLB1 (Imgenex, IMG-265A), guinea pig anti-phospho-SH3GLB1 custom made with synthetic peptide (NFLpTPLRNFIC, PSL), rabbit anti-RAB5 (Cell Signalling Technology, 3547), mouse anti-GAPDH/glyceraldehyde-3-phosphate dehydrogenase (Thermo Fisher Scientific, MA5-15738), rabbit anti-ADRB2/adrenoceptor β 2 (Santa Cruz Biotechnology, sc-569), mouse anti-CHRN (BD Bioscience, 610989), rabbit anti-MAP1LC3B (Genetex, GTX127375), Living Colors® A.v. monoclonal antibody JL-8 (Clontech Laboratories, 632380), anti-mCherry (Acris, AP32117 PU), anti-USP6NL (Bethyl Laboratories, A302-794A-M), anti-RABGEF1 (Cell Signalling Technology, 7622), anti-rabbit coupled to horseradish peroxidase (HRP; Dako, P0448), anti-mouse-HRP (Thermo Fisher Scientific, 32430), anti-guinea pig-HRP (Thermo Fisher Scientific, PA1-28597), anti goat-HRP (Dako, P0449).

    Western Blot:

    Article Title: Progress of endocytic CHRN to autophagic degradation is regulated by RAB5-GTPase and T145 phosphorylation of SH3GLB1 at mouse neuromuscular junctions in vivo
    Article Snippet: Lysate dots were applied on PVDF membrane followed by immunodetection. .. For affinity coprecipitation ( ) gastrocnemius muscles were injected with 125 pmoles of BGT coupled with biotin (Invitrogen, B1196) and then harvested after 5 h. Tissue lysis was performed as mentioned above, while affinity precipitation with NeutrAvidin beads (Thermo Fisher Scientific, 29202) and western blot were performed as previously described. .. The following antibodies were used for the detection of their respective proteins: mouse anti-SH3GLB1 (Imgenex, IMG-265A), guinea pig anti-phospho-SH3GLB1 custom made with synthetic peptide (NFLpTPLRNFIC, PSL), rabbit anti-RAB5 (Cell Signalling Technology, 3547), mouse anti-GAPDH/glyceraldehyde-3-phosphate dehydrogenase (Thermo Fisher Scientific, MA5-15738), rabbit anti-ADRB2/adrenoceptor β 2 (Santa Cruz Biotechnology, sc-569), mouse anti-CHRN (BD Bioscience, 610989), rabbit anti-MAP1LC3B (Genetex, GTX127375), Living Colors® A.v. monoclonal antibody JL-8 (Clontech Laboratories, 632380), anti-mCherry (Acris, AP32117 PU), anti-USP6NL (Bethyl Laboratories, A302-794A-M), anti-RABGEF1 (Cell Signalling Technology, 7622), anti-rabbit coupled to horseradish peroxidase (HRP; Dako, P0448), anti-mouse-HRP (Thermo Fisher Scientific, 32430), anti-guinea pig-HRP (Thermo Fisher Scientific, PA1-28597), anti goat-HRP (Dako, P0449).

    Article Title: Dynamic trafficking and turnover of JAM-C is essential for endothelial cell migration
    Article Snippet: The lysate was centrifuged at 21,000 × g for 15 min at 4°C and protein concentration determined (Pierce 660 nm Protein Assay Reagent, Thermo Fisher Scientific). .. Eight point five μg lysate was kept for western blot analysis, and 1.6–1.8 mg total protein was added to 250 μl washed high-capacity neutravidin beads (Life Technologies) in low binding tubes (Life Technologies) and rotated overnight at 4°C. .. The beads were washed in 25 mM ammonium bicarbonate buffer, centrifuged at 21,000 × g , and frozen at −80°C before mass spectrometry analysis.

    Article Title: Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells
    Article Snippet: Surface membrane biotinylation FDB muscles were dissected from WT and HTZ mice, stabilized during 1 h at room temperature in Tyrode solution, bubbled with a mixture of 5% CO2 and 95% O2 , and ex vivo stimulated with 0.1 µM insulin during 30 min. Then, muscles were exposed to 1 mg/ml of EZ link Sulfo-NHS-SS-biotin (Thermo Scientific #21331) in Tyrode solution at 4 °C for 60 min. Biotinylation was quenched with 100 mM of cold glycine in Tyrode solution; muscles were washed three times with ice-cold Tyrode, frozen and pulverized in liquid nitrogen and then lysed in ice-cold lysis buffer (mM: 1 EDTA, 50 NaCl, 20 TrisHCl, pH 7.5 plus 1% TX-100 and 0.1% SDS). .. Homogenates were centrifuged at 14.000 g for 10 min and supernatants were mixed with 50 µl of streptavidin-agarose beads (Thermo Scientific #29204) overnight at 4 °C with gentle agitation and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were diluted in loading buffer, subjected to SDS-PAGE and western blot to evaluate GLUT4 and GAPDH expression using specific antibodies (rabbit-polyclonal-anti GLUT4, Abcam ab654 and mouse-monoclonal-anti-GAPDH, Abcam ab9484, respectively). .. GLUT4 immunolabeling in human skeletal muscle biopsies Human open muscle biopsies from two patients carrying the CNM- dynamin-2 mutation p.R465W, one patient carrying the CNM-dynamin-2 mutation p.R369Q, one patient with dystrophy harboring a mutation in dysferlin (dysferlinopathy), one patient with dystrophy harboring a mutation in dystrophin (dystrophinopathy) and one healthy control muscle were performed at the Centre de Référence de Pathologie Neuromusculaire Paris-Est, Institut de Myologie, GHU Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, GH Pitié-Salpêtrière, Paris, France, following written informed consent specially dedicated for diagnosis and research.

    Binding Assay:

    Article Title: Dynamic trafficking and turnover of JAM-C is essential for endothelial cell migration
    Article Snippet: The lysate was centrifuged at 21,000 × g for 15 min at 4°C and protein concentration determined (Pierce 660 nm Protein Assay Reagent, Thermo Fisher Scientific). .. Eight point five μg lysate was kept for western blot analysis, and 1.6–1.8 mg total protein was added to 250 μl washed high-capacity neutravidin beads (Life Technologies) in low binding tubes (Life Technologies) and rotated overnight at 4°C. .. The beads were washed in 25 mM ammonium bicarbonate buffer, centrifuged at 21,000 × g , and frozen at −80°C before mass spectrometry analysis.

    Incubation:

    Article Title: Cellular mechanisms and behavioral consequences of Kv1.2 regulation in the rat cerebellum
    Article Snippet: Biotinylated cerebellar slices were individually sonicated in lysis buffer (50 mM TRIS, 150 mM NaCl, 1 mM EDTA, 0.25% deoxycholate, 1% NP40, 10% glycerol, mammalian protease inhibitor cocktail (Sigma), pH 8.0), and the lysate was then clarified by centrifugation. .. A portion of clarified “Total” lysate was reserved and the remainder was incubated with high-capacity neutravidin bead slurry (Pierce). .. Neutravidin beads and bound biotinylated proteins were isolated by centrifugation and washed repeatedly with lysis buffer.

    Article Title: Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation
    Article Snippet: After pelleting at 10,000 × g, clarified supernatants were incubated with 30μl high-capacity neutravidin agarose beads overnight to capture biotinylated proteins (Thermo Scientific, catalogue number 29204). .. After pelleting at 10,000 × g, clarified supernatants were incubated with 30μl high-capacity neutravidin agarose beads overnight to capture biotinylated proteins (Thermo Scientific, catalogue number 29204). .. In all cases, 50% of the bead eluate and 1% lysate was loaded onto SDS-PAGE gels.

    Article Title: Small GTPase Rab11b regulates degradation of surface membrane L-type Cav1.2 channels
    Article Snippet: .. Soluble protein (1–1.5 mg) was incubated with 75 μl high capacity NeutrAvidin agarose beads (Thermo Scientific Pierce Protein Research Products) for 2 h at 4°C. .. Protein G or NeutrAvidin agarose beads were washed, and proteins were eluted by heating to 95°C for 5 min in SDS-PAGE sample buffer.

    SDS Page:

    Article Title: Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells
    Article Snippet: Surface membrane biotinylation FDB muscles were dissected from WT and HTZ mice, stabilized during 1 h at room temperature in Tyrode solution, bubbled with a mixture of 5% CO2 and 95% O2 , and ex vivo stimulated with 0.1 µM insulin during 30 min. Then, muscles were exposed to 1 mg/ml of EZ link Sulfo-NHS-SS-biotin (Thermo Scientific #21331) in Tyrode solution at 4 °C for 60 min. Biotinylation was quenched with 100 mM of cold glycine in Tyrode solution; muscles were washed three times with ice-cold Tyrode, frozen and pulverized in liquid nitrogen and then lysed in ice-cold lysis buffer (mM: 1 EDTA, 50 NaCl, 20 TrisHCl, pH 7.5 plus 1% TX-100 and 0.1% SDS). .. Homogenates were centrifuged at 14.000 g for 10 min and supernatants were mixed with 50 µl of streptavidin-agarose beads (Thermo Scientific #29204) overnight at 4 °C with gentle agitation and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were diluted in loading buffer, subjected to SDS-PAGE and western blot to evaluate GLUT4 and GAPDH expression using specific antibodies (rabbit-polyclonal-anti GLUT4, Abcam ab654 and mouse-monoclonal-anti-GAPDH, Abcam ab9484, respectively). .. GLUT4 immunolabeling in human skeletal muscle biopsies Human open muscle biopsies from two patients carrying the CNM- dynamin-2 mutation p.R465W, one patient carrying the CNM-dynamin-2 mutation p.R369Q, one patient with dystrophy harboring a mutation in dysferlin (dysferlinopathy), one patient with dystrophy harboring a mutation in dystrophin (dystrophinopathy) and one healthy control muscle were performed at the Centre de Référence de Pathologie Neuromusculaire Paris-Est, Institut de Myologie, GHU Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, GH Pitié-Salpêtrière, Paris, France, following written informed consent specially dedicated for diagnosis and research.

    Expressing:

    Article Title: Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells
    Article Snippet: Surface membrane biotinylation FDB muscles were dissected from WT and HTZ mice, stabilized during 1 h at room temperature in Tyrode solution, bubbled with a mixture of 5% CO2 and 95% O2 , and ex vivo stimulated with 0.1 µM insulin during 30 min. Then, muscles were exposed to 1 mg/ml of EZ link Sulfo-NHS-SS-biotin (Thermo Scientific #21331) in Tyrode solution at 4 °C for 60 min. Biotinylation was quenched with 100 mM of cold glycine in Tyrode solution; muscles were washed three times with ice-cold Tyrode, frozen and pulverized in liquid nitrogen and then lysed in ice-cold lysis buffer (mM: 1 EDTA, 50 NaCl, 20 TrisHCl, pH 7.5 plus 1% TX-100 and 0.1% SDS). .. Homogenates were centrifuged at 14.000 g for 10 min and supernatants were mixed with 50 µl of streptavidin-agarose beads (Thermo Scientific #29204) overnight at 4 °C with gentle agitation and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were diluted in loading buffer, subjected to SDS-PAGE and western blot to evaluate GLUT4 and GAPDH expression using specific antibodies (rabbit-polyclonal-anti GLUT4, Abcam ab654 and mouse-monoclonal-anti-GAPDH, Abcam ab9484, respectively). .. GLUT4 immunolabeling in human skeletal muscle biopsies Human open muscle biopsies from two patients carrying the CNM- dynamin-2 mutation p.R465W, one patient carrying the CNM-dynamin-2 mutation p.R369Q, one patient with dystrophy harboring a mutation in dysferlin (dysferlinopathy), one patient with dystrophy harboring a mutation in dystrophin (dystrophinopathy) and one healthy control muscle were performed at the Centre de Référence de Pathologie Neuromusculaire Paris-Est, Institut de Myologie, GHU Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, GH Pitié-Salpêtrière, Paris, France, following written informed consent specially dedicated for diagnosis and research.

    Magnetic Beads:

    Article Title: Selective enrichment and direct analysis of protein S-palmitoylation sites
    Article Snippet: .. Anti-HA magnetic beads, Invitrosol, High-capacity neutravidin beads were obtained from Thermo Scientific Pierce. .. Mass spectrometry grade trypsin and chymotrypsin were purchased from Promega.

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  • 99
    Thermo Fisher streptavidin coated magnetic beads
    Dual-aptamer detection schematic Thrombin and PDGF-BB are represented by triangles and double-ellipses, respectively. The sensing aptamers (MB-T, MB-P) containing thrombin aptamer (Apt 1A) and PDGF-BB aptamer (Apt 2A) are each labeled with an MB tag (MB1 and MB2) and flanked by a pair of universal primers (5′p and 3′p). Capture aptamers (Bio-T and Bio-P) containing thrombin aptamer (Apt 1B) and PDGF-BB aptamer (Apt 2B) are biotinylated to be attached to <t>streptavidin-coated</t> magnetic beads. Oligo(dT)s are used as the spacer between the aptamer sequences and the beads. After separation, eluted sensing aptamers are amplified by multiplex PCR and quantified.
    Streptavidin Coated Magnetic Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin coated magnetic beads/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin coated magnetic beads - by Bioz Stars, 2021-07
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    95
    Thermo Fisher streptavidin agarose beads
    Insulin-induced translocation of GLUT4 is disrupted in muscle fibers isolated from HTZ mice. ( a–c ) Freshly dissected FDB muscles from 2 month-old WT and HTZ mice were digested with collagenase. Isolated fibers were stimulated during 15 min with 0.1 µM insulin to induce GLUT4 translocation, fixed and immunolabeled with a polyclonal-GLUT4 antibody. Translocation of GLUT4 was estimated by measuring the total intensity fluorescence of GLUT4 in ROIs at the sarcolemma. ( a ) Examples of the ROIs used are drawn in white. GLUT4 was measured on both edges of the confocal image and then averaged. ( b ) Examples images of GLUT4 signal in WT and HTZ fibers at the resting (left panels) and insulin-stimulated condition (right panels). Scale bar = 20 µm. ( c ) The graph show the averaged GLUT4 signal in sarcolemma. Note that insulin-induced GLUT4 translocation is significantly reduced in HTZ myofibers compared to WT myofibers. Data are expressed as mean GLUT4 fluorescence signal ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbols * and # denote significance with respect to WT-resting and WT-insulin-stimulated fibers, respectively. N is between 34 and 66 fibers from at least 10 different animals per genotype. ( d ) FDB muscles were dissected from WT and HTZ mice, stabilized in Tyrode solution, stimulated for 30 min with 0.1 µM insulin and then exposed to 1 mg/ml of biotin at 4 °C during 60 min. After quenching with 100 mM glycine, muscles were frozen and pulverized in liquid nitrogen, lysed and centrifuged at 14.000 g for 10 min. Supernatants were mixed with <t>streptavidin-agarose</t> beads overnight at 4 °C and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were used to evaluate GLUT4 expression by western blot. GAPDH was used as a control that only surface proteins were labeled in biotinylated fractions. On the left are shown representative blots per each condition, on the right are plotted the percentages of GLUT4 in biotinylated fractions. Data are expressed as mean GLUT4% ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbol * denote significance with respect to WT-muscles. N is five different animals per genotype.
    Streptavidin Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin agarose beads/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin agarose beads - by Bioz Stars, 2021-07
    95/100 stars
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    97
    Thermo Fisher streptavidin coupled agarose beads
    Notch1 S2 cleavage occurs at the cell surface. A, upper panel, Myc immunoblot of surface-biotinylated and <t>streptavidin-precipitated</t> U2OS cells transfected with the active LNR CC → SS 6Myc. Left upper panel shows input, and right panel streptavidin
    Streptavidin Coupled Agarose Beads, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin coupled agarose beads/product/Thermo Fisher
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin coupled agarose beads - by Bioz Stars, 2021-07
    97/100 stars
      Buy from Supplier

    Image Search Results


    Dual-aptamer detection schematic Thrombin and PDGF-BB are represented by triangles and double-ellipses, respectively. The sensing aptamers (MB-T, MB-P) containing thrombin aptamer (Apt 1A) and PDGF-BB aptamer (Apt 2A) are each labeled with an MB tag (MB1 and MB2) and flanked by a pair of universal primers (5′p and 3′p). Capture aptamers (Bio-T and Bio-P) containing thrombin aptamer (Apt 1B) and PDGF-BB aptamer (Apt 2B) are biotinylated to be attached to streptavidin-coated magnetic beads. Oligo(dT)s are used as the spacer between the aptamer sequences and the beads. After separation, eluted sensing aptamers are amplified by multiplex PCR and quantified.

    Journal: Biosensors & bioelectronics

    Article Title: Development of a dual aptamer-based multiplex protein biosensor

    doi: 10.1016/j.bios.2010.04.034

    Figure Lengend Snippet: Dual-aptamer detection schematic Thrombin and PDGF-BB are represented by triangles and double-ellipses, respectively. The sensing aptamers (MB-T, MB-P) containing thrombin aptamer (Apt 1A) and PDGF-BB aptamer (Apt 2A) are each labeled with an MB tag (MB1 and MB2) and flanked by a pair of universal primers (5′p and 3′p). Capture aptamers (Bio-T and Bio-P) containing thrombin aptamer (Apt 1B) and PDGF-BB aptamer (Apt 2B) are biotinylated to be attached to streptavidin-coated magnetic beads. Oligo(dT)s are used as the spacer between the aptamer sequences and the beads. After separation, eluted sensing aptamers are amplified by multiplex PCR and quantified.

    Article Snippet: Binding reactions were incubated at room temperature for 30 min followed by the addition of streptavidin coated magnetic beads (Dynal/Invitrogen, Oslo, Norway) for an additional 30 min at room temperature.

    Techniques: Labeling, Magnetic Beads, Amplification, Multiplex Assay, Polymerase Chain Reaction

    Insulin-induced translocation of GLUT4 is disrupted in muscle fibers isolated from HTZ mice. ( a–c ) Freshly dissected FDB muscles from 2 month-old WT and HTZ mice were digested with collagenase. Isolated fibers were stimulated during 15 min with 0.1 µM insulin to induce GLUT4 translocation, fixed and immunolabeled with a polyclonal-GLUT4 antibody. Translocation of GLUT4 was estimated by measuring the total intensity fluorescence of GLUT4 in ROIs at the sarcolemma. ( a ) Examples of the ROIs used are drawn in white. GLUT4 was measured on both edges of the confocal image and then averaged. ( b ) Examples images of GLUT4 signal in WT and HTZ fibers at the resting (left panels) and insulin-stimulated condition (right panels). Scale bar = 20 µm. ( c ) The graph show the averaged GLUT4 signal in sarcolemma. Note that insulin-induced GLUT4 translocation is significantly reduced in HTZ myofibers compared to WT myofibers. Data are expressed as mean GLUT4 fluorescence signal ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbols * and # denote significance with respect to WT-resting and WT-insulin-stimulated fibers, respectively. N is between 34 and 66 fibers from at least 10 different animals per genotype. ( d ) FDB muscles were dissected from WT and HTZ mice, stabilized in Tyrode solution, stimulated for 30 min with 0.1 µM insulin and then exposed to 1 mg/ml of biotin at 4 °C during 60 min. After quenching with 100 mM glycine, muscles were frozen and pulverized in liquid nitrogen, lysed and centrifuged at 14.000 g for 10 min. Supernatants were mixed with streptavidin-agarose beads overnight at 4 °C and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were used to evaluate GLUT4 expression by western blot. GAPDH was used as a control that only surface proteins were labeled in biotinylated fractions. On the left are shown representative blots per each condition, on the right are plotted the percentages of GLUT4 in biotinylated fractions. Data are expressed as mean GLUT4% ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbol * denote significance with respect to WT-muscles. N is five different animals per genotype.

    Journal: Scientific Reports

    Article Title: Dynamin-2 mutations linked to Centronuclear Myopathy impair actin-dependent trafficking in muscle cells

    doi: 10.1038/s41598-017-04418-w

    Figure Lengend Snippet: Insulin-induced translocation of GLUT4 is disrupted in muscle fibers isolated from HTZ mice. ( a–c ) Freshly dissected FDB muscles from 2 month-old WT and HTZ mice were digested with collagenase. Isolated fibers were stimulated during 15 min with 0.1 µM insulin to induce GLUT4 translocation, fixed and immunolabeled with a polyclonal-GLUT4 antibody. Translocation of GLUT4 was estimated by measuring the total intensity fluorescence of GLUT4 in ROIs at the sarcolemma. ( a ) Examples of the ROIs used are drawn in white. GLUT4 was measured on both edges of the confocal image and then averaged. ( b ) Examples images of GLUT4 signal in WT and HTZ fibers at the resting (left panels) and insulin-stimulated condition (right panels). Scale bar = 20 µm. ( c ) The graph show the averaged GLUT4 signal in sarcolemma. Note that insulin-induced GLUT4 translocation is significantly reduced in HTZ myofibers compared to WT myofibers. Data are expressed as mean GLUT4 fluorescence signal ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbols * and # denote significance with respect to WT-resting and WT-insulin-stimulated fibers, respectively. N is between 34 and 66 fibers from at least 10 different animals per genotype. ( d ) FDB muscles were dissected from WT and HTZ mice, stabilized in Tyrode solution, stimulated for 30 min with 0.1 µM insulin and then exposed to 1 mg/ml of biotin at 4 °C during 60 min. After quenching with 100 mM glycine, muscles were frozen and pulverized in liquid nitrogen, lysed and centrifuged at 14.000 g for 10 min. Supernatants were mixed with streptavidin-agarose beads overnight at 4 °C and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were used to evaluate GLUT4 expression by western blot. GAPDH was used as a control that only surface proteins were labeled in biotinylated fractions. On the left are shown representative blots per each condition, on the right are plotted the percentages of GLUT4 in biotinylated fractions. Data are expressed as mean GLUT4% ± SEM. Statistical comparisons were performed utilizing a two-tail t-test Welch corrected for parametric data. The symbol * denote significance with respect to WT-muscles. N is five different animals per genotype.

    Article Snippet: Homogenates were centrifuged at 14.000 g for 10 min and supernatants were mixed with 50 µl of streptavidin-agarose beads (Thermo Scientific #29204) overnight at 4 °C with gentle agitation and then centrifuged at 14.000 g for 3 min. Biotinylated and non-biotinylated fractions were diluted in loading buffer, subjected to SDS-PAGE and western blot to evaluate GLUT4 and GAPDH expression using specific antibodies (rabbit-polyclonal-anti GLUT4, Abcam ab654 and mouse-monoclonal-anti-GAPDH, Abcam ab9484, respectively).

    Techniques: Translocation Assay, Isolation, Mouse Assay, Immunolabeling, Fluorescence, Expressing, Western Blot, Labeling

    Notch1 S2 cleavage occurs at the cell surface. A, upper panel, Myc immunoblot of surface-biotinylated and streptavidin-precipitated U2OS cells transfected with the active LNR CC → SS 6Myc. Left upper panel shows input, and right panel streptavidin

    Journal: The Journal of Biological Chemistry

    Article Title: Metalloprotease ADAM10 Is Required for Notch1 Site 2 Cleavage *

    doi: 10.1074/jbc.M109.006775

    Figure Lengend Snippet: Notch1 S2 cleavage occurs at the cell surface. A, upper panel, Myc immunoblot of surface-biotinylated and streptavidin-precipitated U2OS cells transfected with the active LNR CC → SS 6Myc. Left upper panel shows input, and right panel streptavidin

    Article Snippet: After input, samples were taken, and streptavidin-coupled agarose beads (Pierce) were added to the supernatant and incubated for 90 min.

    Techniques: Transfection