biotinylated proteins  (Thermo Fisher)


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    Structured Review

    Thermo Fisher biotinylated proteins
    Chemical structures of WP1130 and its derivatives used herein. (A) WP1130, (B) <t>biotinylated</t> WP1130 and an inactive analog.
    Biotinylated Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response"

    Article Title: Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002783

    Chemical structures of WP1130 and its derivatives used herein. (A) WP1130, (B) biotinylated WP1130 and an inactive analog.
    Figure Legend Snippet: Chemical structures of WP1130 and its derivatives used herein. (A) WP1130, (B) biotinylated WP1130 and an inactive analog.

    Techniques Used:

    WP1130 inhibits the host deubiquitinase USP14 in murine macrophages. (A) WP1130 treatment inhibits the activity of multiple DUBs in murine macrophages. RAW cells were treated with DMSO (D, V+D) or 5 µM WP1130 (V+WP) for 30 minutes prior to infection. Cells were then infected with MNV-1 (V+D, V+WP) or mock lysate (D), washed, and incubated for an additional hour. Cell lysates were incubated with a non-hydrolysable ubiquitin conjugated to an HA tag (HA-UbVS) before separation by SDS-PAGE and immunoblotting with an anti-HA antibody. The experiment was performed three times and a representative blot is shown. A band of the anticipated molecular weight for USP14 is indicated by the arrow head. (B) WP1130 treatment inhibits USP14 activity. RAW cells were treated with DMSO (D) or 5 µM WP1130 (WP) and then infected with MNV-1 (MOI 5) or mock lysate, washed, and incubated for an additional hour. Cell lysates were labeled with HA-UbVS and immunoprecipitated using an anti-HA antibody. Proteins were separated by SDS-PAGE and immunoblots performed using an anti-USP14 antibody. A representative blot is shown (top, Active USP14). Densitometry was performed on four independent experiments, quantitated, and normalized to the mock- and DMSO-treated sample (bottom, Quantitation of Active USP14). As a control, immunoblots were performed for total USP14 levels in cell lysates prior to DUB labeling (middle, Total USP14). (C) Biotinylated WP1130 inhibits MNV-1 infection in RAW cells. Cells were treated with DMSO or 5 µM of WP1130 (WP1130), biotinylated WP1130 (Biotin), inactive biotinylated WP1130 analog (Null Biotin) prior to MNV-1 infection (MOI 5). Viral titers were determined by plaque assay 8 hours post-infection. Data from three independent experiments with two experimental replicates per condition are presented as means +/− S.E.M. ** P
    Figure Legend Snippet: WP1130 inhibits the host deubiquitinase USP14 in murine macrophages. (A) WP1130 treatment inhibits the activity of multiple DUBs in murine macrophages. RAW cells were treated with DMSO (D, V+D) or 5 µM WP1130 (V+WP) for 30 minutes prior to infection. Cells were then infected with MNV-1 (V+D, V+WP) or mock lysate (D), washed, and incubated for an additional hour. Cell lysates were incubated with a non-hydrolysable ubiquitin conjugated to an HA tag (HA-UbVS) before separation by SDS-PAGE and immunoblotting with an anti-HA antibody. The experiment was performed three times and a representative blot is shown. A band of the anticipated molecular weight for USP14 is indicated by the arrow head. (B) WP1130 treatment inhibits USP14 activity. RAW cells were treated with DMSO (D) or 5 µM WP1130 (WP) and then infected with MNV-1 (MOI 5) or mock lysate, washed, and incubated for an additional hour. Cell lysates were labeled with HA-UbVS and immunoprecipitated using an anti-HA antibody. Proteins were separated by SDS-PAGE and immunoblots performed using an anti-USP14 antibody. A representative blot is shown (top, Active USP14). Densitometry was performed on four independent experiments, quantitated, and normalized to the mock- and DMSO-treated sample (bottom, Quantitation of Active USP14). As a control, immunoblots were performed for total USP14 levels in cell lysates prior to DUB labeling (middle, Total USP14). (C) Biotinylated WP1130 inhibits MNV-1 infection in RAW cells. Cells were treated with DMSO or 5 µM of WP1130 (WP1130), biotinylated WP1130 (Biotin), inactive biotinylated WP1130 analog (Null Biotin) prior to MNV-1 infection (MOI 5). Viral titers were determined by plaque assay 8 hours post-infection. Data from three independent experiments with two experimental replicates per condition are presented as means +/− S.E.M. ** P

    Techniques Used: Activity Assay, Infection, Incubation, SDS Page, Molecular Weight, Labeling, Immunoprecipitation, Western Blot, Quantitation Assay, Plaque Assay

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006418

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

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

    3) Product Images from "Calcium-Sensing Receptor and Aquaporin 2 Interplay in Hypercalciuria-Associated Renal Concentrating Defect in Humans. An In Vivo and In Vitro Study"

    Article Title: Calcium-Sensing Receptor and Aquaporin 2 Interplay in Hypercalciuria-Associated Renal Concentrating Defect in Humans. An In Vivo and In Vitro Study

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033145

    Effect of ATP stimulation on AQP2 trafficking in MCD4 cells. (A) MCD4 cells were preincubated with 100 µM ATP or used under control conditions and then stimulated with FK 10 −4 M. The amount of apical AQP2 was quantitated by apical surface biotinylation. ATP caused AQP2 membrane accumulation comparable to that found in FK treated cells. The total amount of AQP2 in the starting preparation was comparable in each experimental condition (total AQP2). (B) Densitometric analysis of the 29 kDa biotinylated AQP2 band. Results are expressed as mean values ± S.E.M. The values obtained in three independent experiments are expressed as percentages of the basal condition. Data were compared by one-way Anova and Tukey's multiple comparison test (* P
    Figure Legend Snippet: Effect of ATP stimulation on AQP2 trafficking in MCD4 cells. (A) MCD4 cells were preincubated with 100 µM ATP or used under control conditions and then stimulated with FK 10 −4 M. The amount of apical AQP2 was quantitated by apical surface biotinylation. ATP caused AQP2 membrane accumulation comparable to that found in FK treated cells. The total amount of AQP2 in the starting preparation was comparable in each experimental condition (total AQP2). (B) Densitometric analysis of the 29 kDa biotinylated AQP2 band. Results are expressed as mean values ± S.E.M. The values obtained in three independent experiments are expressed as percentages of the basal condition. Data were compared by one-way Anova and Tukey's multiple comparison test (* P

    Techniques Used:

    Effect of CaR signaling on AQP2 trafficking in MCD4 cells. Apical surface biotinylation. (A) MCD4 cells were preincubated with 5 mM Ca 2+ , 300 µM Gd 3+ or 10 µM NPS-R 568 then exposed to FK10 −4 M or left under control conditions. Apical membrane-expressed AQP2 was quantitated by apical surface biotinylation. FK-induced AQP2 membrane accumulation was significantly reduced in the presence of CaR agonists. CaR agonists induced a mild increase in AQP2 membrane expression even in the absence of FK stimulation. The total amount of AQP2 in the starting preparation was comparable in each experimental condition (total AQP2). (B) Densitometric analysis of the 29 kDa biotinylated AQP2 band. Results are expressed as mean values ± SEM. The values obtained in five independent experiments are expressed as percentages of the basal condition. Data were compared by one-way Anova and Tukey's multiple comparison test (* P
    Figure Legend Snippet: Effect of CaR signaling on AQP2 trafficking in MCD4 cells. Apical surface biotinylation. (A) MCD4 cells were preincubated with 5 mM Ca 2+ , 300 µM Gd 3+ or 10 µM NPS-R 568 then exposed to FK10 −4 M or left under control conditions. Apical membrane-expressed AQP2 was quantitated by apical surface biotinylation. FK-induced AQP2 membrane accumulation was significantly reduced in the presence of CaR agonists. CaR agonists induced a mild increase in AQP2 membrane expression even in the absence of FK stimulation. The total amount of AQP2 in the starting preparation was comparable in each experimental condition (total AQP2). (B) Densitometric analysis of the 29 kDa biotinylated AQP2 band. Results are expressed as mean values ± SEM. The values obtained in five independent experiments are expressed as percentages of the basal condition. Data were compared by one-way Anova and Tukey's multiple comparison test (* P

    Techniques Used: Expressing

    4) Product Images from "Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein"

    Article Title: Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

    Journal: Retrovirology

    doi: 10.1186/1742-4690-9-17

    Mapping of the Ank GAG -1D4 binding site on HIV-1 Gag CA domain . The binding activity of biotinylated Ank GAG 1D4 protein was tested in ELISA towards surface-immobilized lysates of mock-infected Sf9 cells, or baculovirus-infected Sf9 cells expressing recombinant H 6 MA-CA, H 6 CA, Gag amb 241, or Gag amb 276 protein. The quantity of bound Ank GAG 1D4 was assayed by reaction with streptavidin-HRP. Data presented are from triplicate experiments (m ± SEM).
    Figure Legend Snippet: Mapping of the Ank GAG -1D4 binding site on HIV-1 Gag CA domain . The binding activity of biotinylated Ank GAG 1D4 protein was tested in ELISA towards surface-immobilized lysates of mock-infected Sf9 cells, or baculovirus-infected Sf9 cells expressing recombinant H 6 MA-CA, H 6 CA, Gag amb 241, or Gag amb 276 protein. The quantity of bound Ank GAG 1D4 was assayed by reaction with streptavidin-HRP. Data presented are from triplicate experiments (m ± SEM).

    Techniques Used: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Recombinant

    Gag-binding activity of artificial ankyrins . (A) , Competition ELISA . Samples of biotinylated Gag-binders Ank GAG 1B8, Ank GAG 1D4 and Ank GAG 6B4, and of control biotinylated αRep-A3-binder Ank A3 2D3 were mixed with their corresponding non-biotinylated form (black bars), or mixed with irrelevant soluble target (grey bars), or mixed with buffer containing no inhibitor (white bars). Mixtures were added to H 6 MA-CA- or αRep-A3-coated wells, as indicated at the bottom of the panel. Bound-ankyrins were detected by addition of HRP-conjugated extravidin, followed by the TMB substrate. (B) , Far Western blotting . Lysates of BV-H 6 MA-CA-infected Sf9 cells were electrophoresed in SDS-gel, proteins transferred to a PVDF membrane, and membrane cut into strips. Gag-binding activity was determined by incubation of the strips with the different biotinylated ankyrins Ank GAG 1B8, Ank GAG 1D4, Ank GAG 6B4, and Ank A3 2D3, as indicated on top of the strips. On the rightmost strip, the respective positions of the Gag proteins H 6 MA-CA and H 6 MA were determined using anti-histidine tag antibody (arrowheads). (C) , Indirect ELISA . H 6 CA was captured on nickel-coated plate, and used as substrate for binding assay of biotinylated ankyrins Ank GAG 1B8, Ank GAG 1D4, Ank GAG 6B4, and Ank A3 2D3. Bound-ankyrins were quantitated as in (A) . BG, background signal.
    Figure Legend Snippet: Gag-binding activity of artificial ankyrins . (A) , Competition ELISA . Samples of biotinylated Gag-binders Ank GAG 1B8, Ank GAG 1D4 and Ank GAG 6B4, and of control biotinylated αRep-A3-binder Ank A3 2D3 were mixed with their corresponding non-biotinylated form (black bars), or mixed with irrelevant soluble target (grey bars), or mixed with buffer containing no inhibitor (white bars). Mixtures were added to H 6 MA-CA- or αRep-A3-coated wells, as indicated at the bottom of the panel. Bound-ankyrins were detected by addition of HRP-conjugated extravidin, followed by the TMB substrate. (B) , Far Western blotting . Lysates of BV-H 6 MA-CA-infected Sf9 cells were electrophoresed in SDS-gel, proteins transferred to a PVDF membrane, and membrane cut into strips. Gag-binding activity was determined by incubation of the strips with the different biotinylated ankyrins Ank GAG 1B8, Ank GAG 1D4, Ank GAG 6B4, and Ank A3 2D3, as indicated on top of the strips. On the rightmost strip, the respective positions of the Gag proteins H 6 MA-CA and H 6 MA were determined using anti-histidine tag antibody (arrowheads). (C) , Indirect ELISA . H 6 CA was captured on nickel-coated plate, and used as substrate for binding assay of biotinylated ankyrins Ank GAG 1B8, Ank GAG 1D4, Ank GAG 6B4, and Ank A3 2D3. Bound-ankyrins were quantitated as in (A) . BG, background signal.

    Techniques Used: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Far Western Blot, Infection, SDS-Gel, Incubation, Stripping Membranes, Indirect ELISA

    5) Product Images from "Dysbindin Promotes the Post-Endocytic Sorting of G Protein-Coupled Receptors to Lysosomes"

    Article Title: Dysbindin Promotes the Post-Endocytic Sorting of G Protein-Coupled Receptors to Lysosomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009325

    Dysbindin knockdown specifically inhibits proteolysis of internalized D2 receptors without detectably inhibiting receptor internalization. A. Flow cytometric analysis of dopamine-induced internalization of D2 dopamine receptors. Stably transfected HEK293 cells expressing FLAG-D2R, exposed for the indicated time periods to 10 µM dopamine, were analyzed by surface antibody labeling and flow cytometry. Loss of surface receptor immunoreactivity was used to assess ligand-induced internalization in cells transfected with dysbindin siRNA (DYS) and compared to cells transfected with a non-silencing control duplex (CTL). Points represent averaged values (normalized to cells not exposed to dopamine (t = 0) from ≥5 experiments and error bars indicate S.E.M. B. Fractional internalization measured after exposure cells to 10 µM dopamine for 30 min, showing the lack of significant difference between CTL and DYS conditions. C. Surface biotinylation experiment showing that dysbindin knockdown inhibits dopamine-induced proteolysis of FLAG-D2Rs. D. Quantification of time-dependent loss of surface-biotinylated FLAG-D2R in cells incubated for the indicated time period after surface biotinylation in the presence of 10 µM dopamine. E. Comparison of D2R degradation measured at the 4 hour time point over multiple experiments (n = 8), verifying the statistical significance (p = 0.001 by Student's t-test) of the observed inhibition. F. Biotin protection-degradation assay showing that dysbindin knockdown specifically inhibits degradation of D2Rs after internalization.
    Figure Legend Snippet: Dysbindin knockdown specifically inhibits proteolysis of internalized D2 receptors without detectably inhibiting receptor internalization. A. Flow cytometric analysis of dopamine-induced internalization of D2 dopamine receptors. Stably transfected HEK293 cells expressing FLAG-D2R, exposed for the indicated time periods to 10 µM dopamine, were analyzed by surface antibody labeling and flow cytometry. Loss of surface receptor immunoreactivity was used to assess ligand-induced internalization in cells transfected with dysbindin siRNA (DYS) and compared to cells transfected with a non-silencing control duplex (CTL). Points represent averaged values (normalized to cells not exposed to dopamine (t = 0) from ≥5 experiments and error bars indicate S.E.M. B. Fractional internalization measured after exposure cells to 10 µM dopamine for 30 min, showing the lack of significant difference between CTL and DYS conditions. C. Surface biotinylation experiment showing that dysbindin knockdown inhibits dopamine-induced proteolysis of FLAG-D2Rs. D. Quantification of time-dependent loss of surface-biotinylated FLAG-D2R in cells incubated for the indicated time period after surface biotinylation in the presence of 10 µM dopamine. E. Comparison of D2R degradation measured at the 4 hour time point over multiple experiments (n = 8), verifying the statistical significance (p = 0.001 by Student's t-test) of the observed inhibition. F. Biotin protection-degradation assay showing that dysbindin knockdown specifically inhibits degradation of D2Rs after internalization.

    Techniques Used: Flow Cytometry, Stable Transfection, Transfection, Expressing, Antibody Labeling, Cytometry, CTL Assay, Incubation, Inhibition, Degradation Assay

    6) Product Images from "Asp433 in the closing gate of ASIC1 determines stability of the open state without changing properties of the selectivity filter or Ca2+ block"

    Article Title: Asp433 in the closing gate of ASIC1 determines stability of the open state without changing properties of the selectivity filter or Ca2+ block

    Journal: The Journal of General Physiology

    doi: 10.1085/jgp.201010576

    Relative magnitude of whole cell currents evoked by pH 6.0 of mutants in positions Gly-432 and Asp-433. (A) Normalized currents of Gly-432 substitutions. Columns are the average of ≥12 cells from three different preparations. Error bars are standard deviations. Asterisks indicate significant difference from G432 by unpaired t test, P ≤ 0.005. (B) Western blot of lASIC1 detected with anti-FLAG monoclonal antibody from eight oocytes injected with 5 ng cRNA that were biotinylated after measurements of whole cell currents by TEVC. (C) Normalized currents of substitutions of Asp-433. (D) Western blot of oocytes as shown in C, but oocytes expressing N433 and G433 were injected with 50 ng cRNA.
    Figure Legend Snippet: Relative magnitude of whole cell currents evoked by pH 6.0 of mutants in positions Gly-432 and Asp-433. (A) Normalized currents of Gly-432 substitutions. Columns are the average of ≥12 cells from three different preparations. Error bars are standard deviations. Asterisks indicate significant difference from G432 by unpaired t test, P ≤ 0.005. (B) Western blot of lASIC1 detected with anti-FLAG monoclonal antibody from eight oocytes injected with 5 ng cRNA that were biotinylated after measurements of whole cell currents by TEVC. (C) Normalized currents of substitutions of Asp-433. (D) Western blot of oocytes as shown in C, but oocytes expressing N433 and G433 were injected with 50 ng cRNA.

    Techniques Used: Western Blot, Injection, Expressing

    7) Product Images from "Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)"

    Article Title: Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-12-27

    LRRC32 is a cell surface protein . a) C-and N- terminus GFP-tagged LRRC32 expressing HEK293 cell clones were surface biotinylated, and cell lysates were immunoprecipitated using antibody specific for GFP or using normal rabbit serum (NRS) as a control (left and right panels). Protein lysates were then electrophoresed, transferred to membrane PDVF, and probed for the presence of biotinylation using streptavidin-HRP (left panel only). Blots were also probed with anti-GFP (right panel only). b) Confocal analysis of untransfected (top row) HEK293 cells, C-GFP/CAT-transfected HEK293 cells (second row), N-GFP/LRRC32-transfected HEK293 cells (third row), C-GFP/LRRC32-transfected HEK293 cells (fourth row), and C-GFP/LRRC32ΔSP-transfected HEK293 cells (last row); Green = GFP, Red = anti-LRRC32 antibody, Blue = nuclear counterstain. The left column shows anti-LRRC32 only. The right column shows GFP only. The middle column shows the merged composite confocal picture (anti-LRRC32 + GFP) with the nuclear counterstain.
    Figure Legend Snippet: LRRC32 is a cell surface protein . a) C-and N- terminus GFP-tagged LRRC32 expressing HEK293 cell clones were surface biotinylated, and cell lysates were immunoprecipitated using antibody specific for GFP or using normal rabbit serum (NRS) as a control (left and right panels). Protein lysates were then electrophoresed, transferred to membrane PDVF, and probed for the presence of biotinylation using streptavidin-HRP (left panel only). Blots were also probed with anti-GFP (right panel only). b) Confocal analysis of untransfected (top row) HEK293 cells, C-GFP/CAT-transfected HEK293 cells (second row), N-GFP/LRRC32-transfected HEK293 cells (third row), C-GFP/LRRC32-transfected HEK293 cells (fourth row), and C-GFP/LRRC32ΔSP-transfected HEK293 cells (last row); Green = GFP, Red = anti-LRRC32 antibody, Blue = nuclear counterstain. The left column shows anti-LRRC32 only. The right column shows GFP only. The middle column shows the merged composite confocal picture (anti-LRRC32 + GFP) with the nuclear counterstain.

    Techniques Used: Expressing, Clone Assay, Immunoprecipitation, Transfection

    8) Product Images from "Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions"

    Article Title: Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1001025

    Local [Ca 2+ ] i determines plasma membrane insertion of TRPC1. (A) Tg treatment and biotinylation were carried out with HSG cells loaded with 200 µM BAPTA-AM (as described in Materials and Methods ), and quantitation of TRPC1 in the fraction pulled down with avidin is shown on the right. The change induced by Tg is not significantly less than that seen in control cells in Figure 3 . (B) Tg-stimulated increase in fura-2 fluorescence was measured in HSG cells loaded with either 50 µM or 200 µM BAPTA-AM. (C) Biotinylation of TRPC1 in cells treated under the conditions indicated in the figure. Levels of TRPC1 and Orai1 in the cell lysates are shown in the blot. (D) Quantitation of blots from four similar experiments as shown in (C) depict the increase in TRPC1 in the biotinylated fraction relative to control. ** indicates values significantly different from unmarked values but not from each other. (E, F) Effect of extracellular Ca 2+ on detection of I SOC . HSG cells were either incubated for 10–15 min with Tg in nominally Ca 2+ -free medium (E) or Ca 2+ -containing medium (F). Cells were then patched in the Ca 2+ -free condition and then switched to DVF medium. In each case, current developments are shown by traces on the left and I–V curves of the current measured at the indicated time points are given on the right.
    Figure Legend Snippet: Local [Ca 2+ ] i determines plasma membrane insertion of TRPC1. (A) Tg treatment and biotinylation were carried out with HSG cells loaded with 200 µM BAPTA-AM (as described in Materials and Methods ), and quantitation of TRPC1 in the fraction pulled down with avidin is shown on the right. The change induced by Tg is not significantly less than that seen in control cells in Figure 3 . (B) Tg-stimulated increase in fura-2 fluorescence was measured in HSG cells loaded with either 50 µM or 200 µM BAPTA-AM. (C) Biotinylation of TRPC1 in cells treated under the conditions indicated in the figure. Levels of TRPC1 and Orai1 in the cell lysates are shown in the blot. (D) Quantitation of blots from four similar experiments as shown in (C) depict the increase in TRPC1 in the biotinylated fraction relative to control. ** indicates values significantly different from unmarked values but not from each other. (E, F) Effect of extracellular Ca 2+ on detection of I SOC . HSG cells were either incubated for 10–15 min with Tg in nominally Ca 2+ -free medium (E) or Ca 2+ -containing medium (F). Cells were then patched in the Ca 2+ -free condition and then switched to DVF medium. In each case, current developments are shown by traces on the left and I–V curves of the current measured at the indicated time points are given on the right.

    Techniques Used: Quantitation Assay, Avidin-Biotin Assay, Fluorescence, Incubation

    Orai1-mediated Ca 2+ entry induces plasma membrane insertion of TRPC1 channels. Biotinylation of TRPC1 was determined in HSG cells (see Materials and Methods for details). (A) Detection of TRPC1 in the biotinylated fraction from control HSG cells and HSG cells treated with siOrai1 or expressing Orai1-E106Q in resting conditions or following treatment with Tg (see C for quantitation of the bands and average data from five individual experiments). (B) TRPC1 biotinylation in HSG cells treated with Tg either in the presence of Gd 3+ or in Ca 2+ -free medium (see C for quantitation of data and average values obtained from four experiments). (C) Quantitation of TRPC1 surface expression shown in (A) and (B). ** indicates value significantly, p
    Figure Legend Snippet: Orai1-mediated Ca 2+ entry induces plasma membrane insertion of TRPC1 channels. Biotinylation of TRPC1 was determined in HSG cells (see Materials and Methods for details). (A) Detection of TRPC1 in the biotinylated fraction from control HSG cells and HSG cells treated with siOrai1 or expressing Orai1-E106Q in resting conditions or following treatment with Tg (see C for quantitation of the bands and average data from five individual experiments). (B) TRPC1 biotinylation in HSG cells treated with Tg either in the presence of Gd 3+ or in Ca 2+ -free medium (see C for quantitation of data and average values obtained from four experiments). (C) Quantitation of TRPC1 surface expression shown in (A) and (B). ** indicates value significantly, p

    Techniques Used: Expressing, Quantitation Assay

    9) Product Images from "The Sigma-1 Receptor Binds to the Nav1.5 Voltage-gated Na+ Channel with 4-Fold Symmetry *"

    Article Title: The Sigma-1 Receptor Binds to the Nav1.5 Voltage-gated Na+ Channel with 4-Fold Symmetry *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.382077

    Isolation and analysis of Sig1R-FLAG·Nav1.5-HA complexes by immunoaffinity chromatography on anti-FLAG-agarose. A , samples of protein isolated by affinity chromatography were analyzed by SDS-PAGE followed by immunoblotting using either mouse monoclonal anti-FLAG ( left panel ) or mouse monoclonal anti-HA antibodies ( right panel ). Arrowheads indicate molecular mass markers (kDa). B , a sample of protein isolated from surface-biotinylated, intact co-transfected cells by sequential affinity chromatography on monomeric avidin-agarose and anti-FLAG-agarose was analyzed by SDS-PAGE followed by immunoblotting using either mouse monoclonal anti-FLAG ( bottom panel ) or mouse monoclonal anti-HA antibodies ( top panel ). A crude detergent extract of the cells (at a loading ratio of 1:128 compared with the final eluate) was also analyzed. C , low-magnification AFM images of samples of isolated Sig1R-FLAG/Nav1.5. Singly- and doubly-decorated large particles are indicated by arrowheads and an arrow , respectively. D , frequency distribution of volumes of large particles that were decorated by Sig1R particles. The curve indicates the fitted Gaussian function. The peak of the distribution is indicated. E , gallery of enlarged images of Nav1.5 channels that were decorated by either one ( upper panels ) or two Sig1Rs ( lower panels ). Angles between pairs of bound Sig1Rs are indicated. F , frequency distribution of angles between pairs of bound Sig1Rs. The curve indicates the fitted Gaussian functions. The peaks of the distribution are indicated. G , gallery of zoomed images of Nav1.5 particles decorated by one, two, three, or four Sig1R particles. Angles between pairs of bound Sig1Rs are indicated.
    Figure Legend Snippet: Isolation and analysis of Sig1R-FLAG·Nav1.5-HA complexes by immunoaffinity chromatography on anti-FLAG-agarose. A , samples of protein isolated by affinity chromatography were analyzed by SDS-PAGE followed by immunoblotting using either mouse monoclonal anti-FLAG ( left panel ) or mouse monoclonal anti-HA antibodies ( right panel ). Arrowheads indicate molecular mass markers (kDa). B , a sample of protein isolated from surface-biotinylated, intact co-transfected cells by sequential affinity chromatography on monomeric avidin-agarose and anti-FLAG-agarose was analyzed by SDS-PAGE followed by immunoblotting using either mouse monoclonal anti-FLAG ( bottom panel ) or mouse monoclonal anti-HA antibodies ( top panel ). A crude detergent extract of the cells (at a loading ratio of 1:128 compared with the final eluate) was also analyzed. C , low-magnification AFM images of samples of isolated Sig1R-FLAG/Nav1.5. Singly- and doubly-decorated large particles are indicated by arrowheads and an arrow , respectively. D , frequency distribution of volumes of large particles that were decorated by Sig1R particles. The curve indicates the fitted Gaussian function. The peak of the distribution is indicated. E , gallery of enlarged images of Nav1.5 channels that were decorated by either one ( upper panels ) or two Sig1Rs ( lower panels ). Angles between pairs of bound Sig1Rs are indicated. F , frequency distribution of angles between pairs of bound Sig1Rs. The curve indicates the fitted Gaussian functions. The peaks of the distribution are indicated. G , gallery of zoomed images of Nav1.5 particles decorated by one, two, three, or four Sig1R particles. Angles between pairs of bound Sig1Rs are indicated.

    Techniques Used: Isolation, Chromatography, Affinity Chromatography, SDS Page, Transfection, Avidin-Biotin Assay

    10) Product Images from "The Sigma-1 Receptor Binds to the Nav1.5 Voltage-gated Na+ Channel with 4-Fold Symmetry *"

    Article Title: The Sigma-1 Receptor Binds to the Nav1.5 Voltage-gated Na+ Channel with 4-Fold Symmetry *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.382077

    Isolation and analysis of Sig1R-FLAG·Nav1.5-HA complexes by immunoaffinity chromatography on anti-FLAG-agarose. A , samples of protein isolated by affinity chromatography were analyzed by SDS-PAGE followed by immunoblotting using either mouse monoclonal anti-FLAG ( left panel ) or mouse monoclonal anti-HA antibodies ( right panel ). Arrowheads indicate molecular mass markers (kDa). B , a sample of protein isolated from surface-biotinylated, intact co-transfected cells by sequential affinity chromatography on monomeric avidin-agarose and anti-FLAG-agarose was analyzed by SDS-PAGE followed by immunoblotting using either mouse monoclonal anti-FLAG ( bottom panel ) or mouse monoclonal anti-HA antibodies ( top panel ). A crude detergent extract of the cells (at a loading ratio of 1:128 compared with the final eluate) was also analyzed. C , low-magnification AFM images of samples of isolated Sig1R-FLAG/Nav1.5. Singly- and doubly-decorated large particles are indicated by arrowheads and an arrow , respectively. D , frequency distribution of volumes of large particles that were decorated by Sig1R particles. The curve indicates the fitted Gaussian function. The peak of the distribution is indicated. E , gallery of enlarged images of Nav1.5 channels that were decorated by either one ( upper panels ) or two Sig1Rs ( lower panels ). Angles between pairs of bound Sig1Rs are indicated. F , frequency distribution of angles between pairs of bound Sig1Rs. The curve indicates the fitted Gaussian functions. The peaks of the distribution are indicated. G , gallery of zoomed images of Nav1.5 particles decorated by one, two, three, or four Sig1R particles. Angles between pairs of bound Sig1Rs are indicated.
    Figure Legend Snippet: Isolation and analysis of Sig1R-FLAG·Nav1.5-HA complexes by immunoaffinity chromatography on anti-FLAG-agarose. A , samples of protein isolated by affinity chromatography were analyzed by SDS-PAGE followed by immunoblotting using either mouse monoclonal anti-FLAG ( left panel ) or mouse monoclonal anti-HA antibodies ( right panel ). Arrowheads indicate molecular mass markers (kDa). B , a sample of protein isolated from surface-biotinylated, intact co-transfected cells by sequential affinity chromatography on monomeric avidin-agarose and anti-FLAG-agarose was analyzed by SDS-PAGE followed by immunoblotting using either mouse monoclonal anti-FLAG ( bottom panel ) or mouse monoclonal anti-HA antibodies ( top panel ). A crude detergent extract of the cells (at a loading ratio of 1:128 compared with the final eluate) was also analyzed. C , low-magnification AFM images of samples of isolated Sig1R-FLAG/Nav1.5. Singly- and doubly-decorated large particles are indicated by arrowheads and an arrow , respectively. D , frequency distribution of volumes of large particles that were decorated by Sig1R particles. The curve indicates the fitted Gaussian function. The peak of the distribution is indicated. E , gallery of enlarged images of Nav1.5 channels that were decorated by either one ( upper panels ) or two Sig1Rs ( lower panels ). Angles between pairs of bound Sig1Rs are indicated. F , frequency distribution of angles between pairs of bound Sig1Rs. The curve indicates the fitted Gaussian functions. The peaks of the distribution are indicated. G , gallery of zoomed images of Nav1.5 particles decorated by one, two, three, or four Sig1R particles. Angles between pairs of bound Sig1Rs are indicated.

    Techniques Used: Isolation, Chromatography, Affinity Chromatography, SDS Page, Transfection, Avidin-Biotin Assay

    11) Product Images from "Role of Phosphatidylinositol Clathrin Assembly Lymphoid-Myeloid Leukemia (PICALM) in Intracellular Amyloid Precursor Protein (APP) Processing and Amyloid Plaque Pathogenesis *"

    Article Title: Role of Phosphatidylinositol Clathrin Assembly Lymphoid-Myeloid Leukemia (PICALM) in Intracellular Amyloid Precursor Protein (APP) Processing and Amyloid Plaque Pathogenesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.338376

    PICALM manipulation alters cell surface APP and APP internalization. N2a-APP695 cells were transfected with shPICALM, shScrambled ( shScr ), PICALM-HA, or empty vector. Twenty-four hours after transfection, cell surface proteins were biotinylated at 4 °C.
    Figure Legend Snippet: PICALM manipulation alters cell surface APP and APP internalization. N2a-APP695 cells were transfected with shPICALM, shScrambled ( shScr ), PICALM-HA, or empty vector. Twenty-four hours after transfection, cell surface proteins were biotinylated at 4 °C.

    Techniques Used: Transfection, Plasmid Preparation

    12) Product Images from "?-Taxilin Interacts with Sorting Nexin 4 and Participates in the Recycling Pathway of Transferrin Receptor"

    Article Title: ?-Taxilin Interacts with Sorting Nexin 4 and Participates in the Recycling Pathway of Transferrin Receptor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093509

    Knockdown of α-taxilin impedes the recycling of Tfn. (A) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with sulfo-NHS-SS-biotin at 4°C, and then the cells were incubated at 37°C for the indicated periods of time. Cells were treated with MesNa to remove biotin remaining on the plasma membrane, and then the cell lysates were precipitated with neutravidin-agarose beads. The precipitates were probed with an anti-TfnR antibody (biotinylated TfnR). The cell lysates used for precipitation were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. (B) The amount of internalized TfnR in (A) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of internalized TfnR at the indicated time periods to biotinylated TfnR at time zero without MesNa treatment from three independent experiments. P -values (control cells vs. α-taxilin knockdown cells at 2.5, 5, 10 min) determined by Student's t -test was not significant. (C) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were incubated with Tfn-488 at 37°C for 1 h. In the case of treatment with leupeptin, the cells were preincubated with leupeptin (200 μg/ml) 1 h prior to Tfn-488 labeling. After washing out unbound Tfn-488, the cells were incubated at 37°C for various time periods in the presence or absence of leupeptin (200 μg/ml). Scale bars, 10 μm. (D) The intensity of Tfn-488 signal of HeLaS3 cells untreated with leupeptin in (C) was expressed as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at each time point to Tfn-488 at time zero. Values at time zero are set to 1.0. P -values (control cells vs. α-taxilin knockdown cells at 10, 20, 40 min) are determined by Student's t -test. *, P
    Figure Legend Snippet: Knockdown of α-taxilin impedes the recycling of Tfn. (A) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were treated with sulfo-NHS-SS-biotin at 4°C, and then the cells were incubated at 37°C for the indicated periods of time. Cells were treated with MesNa to remove biotin remaining on the plasma membrane, and then the cell lysates were precipitated with neutravidin-agarose beads. The precipitates were probed with an anti-TfnR antibody (biotinylated TfnR). The cell lysates used for precipitation were probed with anti-TfnR, anti-α-taxilin and anti-clathrin heavy chain antibodies. The results shown are representative of three independent experiments. (B) The amount of internalized TfnR in (A) was quantified using Image J software. The results shown are means ± s.e.m. of the ratio of internalized TfnR at the indicated time periods to biotinylated TfnR at time zero without MesNa treatment from three independent experiments. P -values (control cells vs. α-taxilin knockdown cells at 2.5, 5, 10 min) determined by Student's t -test was not significant. (C) HeLaS3 cells transfected with control or α-taxilin siRNA (#3) were serum starved for 3 h, and then the cells were incubated with Tfn-488 at 37°C for 1 h. In the case of treatment with leupeptin, the cells were preincubated with leupeptin (200 μg/ml) 1 h prior to Tfn-488 labeling. After washing out unbound Tfn-488, the cells were incubated at 37°C for various time periods in the presence or absence of leupeptin (200 μg/ml). Scale bars, 10 μm. (D) The intensity of Tfn-488 signal of HeLaS3 cells untreated with leupeptin in (C) was expressed as signal intensity per unit area. At each time point, signal intensity of at least 20 cells was measured from three independent experiments. The results shown are means ± s.e.m. of the ratio of Tfn-488 at each time point to Tfn-488 at time zero. Values at time zero are set to 1.0. P -values (control cells vs. α-taxilin knockdown cells at 10, 20, 40 min) are determined by Student's t -test. *, P

    Techniques Used: Transfection, Incubation, Software, Labeling

    13) Product Images from "An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations"

    Article Title: An AP-MS- and BioID-compatible MAC-tag enables comprehensive mapping of protein interactions and subcellular localizations

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03523-2

    Fluorescence microscopy analysis of the cellular localization markers. The 18 subcellular localization markers fused with MAC-tag were visualized by immunofluorescence staining using Alexa Fluor 488 labeled anti-HA immunostaining (green), their in vivo biotinylated interactors with Alexa Fluor 594 streptavidin (red), and cell nuclei with DAPI (blue) (Scale bar: 10 μm)
    Figure Legend Snippet: Fluorescence microscopy analysis of the cellular localization markers. The 18 subcellular localization markers fused with MAC-tag were visualized by immunofluorescence staining using Alexa Fluor 488 labeled anti-HA immunostaining (green), their in vivo biotinylated interactors with Alexa Fluor 594 streptavidin (red), and cell nuclei with DAPI (blue) (Scale bar: 10 μm)

    Techniques Used: Fluorescence, Microscopy, Immunofluorescence, Staining, Labeling, Immunostaining, In Vivo

    14) Product Images from "BK channel blocker paxilline attenuates thalidomide-caused synaptic and cognitive dysfunctions in mice"

    Article Title: BK channel blocker paxilline attenuates thalidomide-caused synaptic and cognitive dysfunctions in mice

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36367-3

    Thalidomide treatment increases BK channel activity and reduces presynaptic glutamate release probability. ( a ) Surface expression of BK channels increased in thalidomide-treated hippocampal slices. Hippocampal slices prepared from vehicle (Veh) and thalidomide (Thal)-treated mice were used for steady-state biotinylation of surface BK channels. Input (25%) of total lysates are shown in the bottom panel, and the biotinylated surface BK channels are shown in the top panel. The quantification of biotinylated BK channels and total BK channels is presented as mean ± SEM. n = 6. ( b ) The increase in calcium-activated potassium currents (I K(Ca) ) in hippocampal CA1 pyramidal neurons due to a 3 h incubation with 100 μM thalidomide was reduced by bath-application of 10 μM paxilline or 100 nM iberiotoxin. I K(Ca) was evoked by brief depolarization from the holding potential (−50 mV) under a TTX-including external solution. n = 32 slices, 9 mice (vehicle); n = 42, 7 (thalidomide); n = 17, 5 (paxilline); n = 19, 5 (thalidomide + paxilline); n = 12, 4 (iberiotoxin); n = 12, 4 (thalidomide + iberiotoxin). ( c ) Paxilline (10 μM) abolishes the increased paired-pulse ratio (PPR, 50 ms inter stimulus interval) at SC-CA1 synapses in mouse hippocampal slices initially incubated with 100 μM thalidomide for at least 3 h. n = 22, 5 (vehicle); n = 19, 6 (thalidomide); n = 29, 4 (paxilline); n = 20, 3 (thalidomide + paxilline). # P
    Figure Legend Snippet: Thalidomide treatment increases BK channel activity and reduces presynaptic glutamate release probability. ( a ) Surface expression of BK channels increased in thalidomide-treated hippocampal slices. Hippocampal slices prepared from vehicle (Veh) and thalidomide (Thal)-treated mice were used for steady-state biotinylation of surface BK channels. Input (25%) of total lysates are shown in the bottom panel, and the biotinylated surface BK channels are shown in the top panel. The quantification of biotinylated BK channels and total BK channels is presented as mean ± SEM. n = 6. ( b ) The increase in calcium-activated potassium currents (I K(Ca) ) in hippocampal CA1 pyramidal neurons due to a 3 h incubation with 100 μM thalidomide was reduced by bath-application of 10 μM paxilline or 100 nM iberiotoxin. I K(Ca) was evoked by brief depolarization from the holding potential (−50 mV) under a TTX-including external solution. n = 32 slices, 9 mice (vehicle); n = 42, 7 (thalidomide); n = 17, 5 (paxilline); n = 19, 5 (thalidomide + paxilline); n = 12, 4 (iberiotoxin); n = 12, 4 (thalidomide + iberiotoxin). ( c ) Paxilline (10 μM) abolishes the increased paired-pulse ratio (PPR, 50 ms inter stimulus interval) at SC-CA1 synapses in mouse hippocampal slices initially incubated with 100 μM thalidomide for at least 3 h. n = 22, 5 (vehicle); n = 19, 6 (thalidomide); n = 29, 4 (paxilline); n = 20, 3 (thalidomide + paxilline). # P

    Techniques Used: Activity Assay, Expressing, Mouse Assay, Incubation, Mass Spectrometry

    15) Product Images from "N-glycosylation is essential for ileal ASBT function and protection against proteases"

    Article Title: N-glycosylation is essential for ileal ASBT function and protection against proteases

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00023.2015

    N -glycosylation increases ASBT protein stability. A : HEK-293 cells stably expressing wild-type ASBT or glycosylation deficient N10Q mutant ASBT were treated with 0.1 mg/ml of cycloheximide for different periods of time and then cell surface proteins were biotinylated. The labeled cell surface proteins were precipitated with streptavidin beads and resolved by SDS-PAGE followed by Western blotting with anti-V5 antibodies (UB, upper band; LB, lower band). B : densitometric analysis of surface levels of wild-type ASBT and glycosylation deficient mutant ASBT-N10Q (up triangles, UB; down triangles, LB; circles, N10Q mutant). Values are expressed as % control (values at 0 h).
    Figure Legend Snippet: N -glycosylation increases ASBT protein stability. A : HEK-293 cells stably expressing wild-type ASBT or glycosylation deficient N10Q mutant ASBT were treated with 0.1 mg/ml of cycloheximide for different periods of time and then cell surface proteins were biotinylated. The labeled cell surface proteins were precipitated with streptavidin beads and resolved by SDS-PAGE followed by Western blotting with anti-V5 antibodies (UB, upper band; LB, lower band). B : densitometric analysis of surface levels of wild-type ASBT and glycosylation deficient mutant ASBT-N10Q (up triangles, UB; down triangles, LB; circles, N10Q mutant). Values are expressed as % control (values at 0 h).

    Techniques Used: Stable Transfection, Expressing, Mutagenesis, Labeling, SDS Page, Western Blot

    N -glycosylation is not essential for ASBT trafficking to plasma membrane. A : transiently transfected cells were biotinylated at various time points posttransfection, and cell surface proteins were precipitated with streptavidin beads followed by Western blotting with anti-V5 antibody. UB, upper band; LB, lower band. B : densitometric analysis for the surface expression of wild-type ASBT (up triangles, UB; down triangles, LB) and ASBT-N10Q (circles) at various times posttransfection. Values are expressed as % of maximal expression and represent means ± SE of 3 experiments.
    Figure Legend Snippet: N -glycosylation is not essential for ASBT trafficking to plasma membrane. A : transiently transfected cells were biotinylated at various time points posttransfection, and cell surface proteins were precipitated with streptavidin beads followed by Western blotting with anti-V5 antibody. UB, upper band; LB, lower band. B : densitometric analysis for the surface expression of wild-type ASBT (up triangles, UB; down triangles, LB) and ASBT-N10Q (circles) at various times posttransfection. Values are expressed as % of maximal expression and represent means ± SE of 3 experiments.

    Techniques Used: Transfection, Western Blot, Expressing

    16) Product Images from "Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *"

    Article Title: Mechanism and Function of Monoclonal Antibodies Targeting Siglec-15 for Therapeutic Inhibition of Osteoclastic Bone Resorption *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.494542

    Antibody-induced internalization and lysosomal degradation of Siglec-15 in osteoclasts. A, internalization of biotinylated Siglec-15. RAW264.7-derived osteoclast cell-surface proteins were labeled with a disulfide-linked biotinylation reagent. Cells were then treated with a combination of primary (anti-Siglec-15 B02 or control IgG) antibodies (at 4 °C) followed by secondary cross-linking antibodies (10 min at 37 °C, lanes 5 and 6 ), or with primary antibody alone (10 min at 37 °C, lanes 7 and 8 ). Control cells were incubated with B02, control IgG, or no antibody at 4 °C ( lanes 1–3 ) without a 37 °C chase. After these antibody treatments, remaining cell-surface biotin was stripped with a reducing agent; for one sample, as an additional control ( lane 4 ), this stripping step was omitted. Internalized, biotinylated proteins were collected from cell lysates by streptavidin immunoprecipitation, and Siglec-15 was detected by Western blotting. B, characterization of Siglec-15 endocytosis by confocal microscopy. RAW264.7-derived osteoclasts were cold-loaded with Siglec-15 antibody and either fixed immediately ( No chase ) or incubated in fresh, warm media for 10 or 45 min prior to fixation. Cells were then permeablized and stained with anti-LAMP2 and anti-human IgG (to detect internalized Siglec-15). C, Siglec-15 protein levels decrease rapidly following antibody treatment. Differentiated RAW264.7-derived osteoclasts were treated for the indicated times with anti-Siglec-15 or a control human IgG. Protein lysates were analyzed by Western blotting with the indicated antibodies. D , RAW264.7 differentiated in the presence of Siglec-15 antibodies show decreased Siglec-15 protein levels. Protein lysates of RAW264.7 cells, either non-differentiated (− RANKL ) or differentiated (+ RANKL ) in media with or without anti-Siglec-15 were analyzed by Western blotting with the indicated antibodies.
    Figure Legend Snippet: Antibody-induced internalization and lysosomal degradation of Siglec-15 in osteoclasts. A, internalization of biotinylated Siglec-15. RAW264.7-derived osteoclast cell-surface proteins were labeled with a disulfide-linked biotinylation reagent. Cells were then treated with a combination of primary (anti-Siglec-15 B02 or control IgG) antibodies (at 4 °C) followed by secondary cross-linking antibodies (10 min at 37 °C, lanes 5 and 6 ), or with primary antibody alone (10 min at 37 °C, lanes 7 and 8 ). Control cells were incubated with B02, control IgG, or no antibody at 4 °C ( lanes 1–3 ) without a 37 °C chase. After these antibody treatments, remaining cell-surface biotin was stripped with a reducing agent; for one sample, as an additional control ( lane 4 ), this stripping step was omitted. Internalized, biotinylated proteins were collected from cell lysates by streptavidin immunoprecipitation, and Siglec-15 was detected by Western blotting. B, characterization of Siglec-15 endocytosis by confocal microscopy. RAW264.7-derived osteoclasts were cold-loaded with Siglec-15 antibody and either fixed immediately ( No chase ) or incubated in fresh, warm media for 10 or 45 min prior to fixation. Cells were then permeablized and stained with anti-LAMP2 and anti-human IgG (to detect internalized Siglec-15). C, Siglec-15 protein levels decrease rapidly following antibody treatment. Differentiated RAW264.7-derived osteoclasts were treated for the indicated times with anti-Siglec-15 or a control human IgG. Protein lysates were analyzed by Western blotting with the indicated antibodies. D , RAW264.7 differentiated in the presence of Siglec-15 antibodies show decreased Siglec-15 protein levels. Protein lysates of RAW264.7 cells, either non-differentiated (− RANKL ) or differentiated (+ RANKL ) in media with or without anti-Siglec-15 were analyzed by Western blotting with the indicated antibodies.

    Techniques Used: Derivative Assay, Labeling, Incubation, Stripping Membranes, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining

    17) Product Images from "Dual Processing of FAT1 Cadherin Protein by Human Melanoma Cells Generates Distinct Protein Products *"

    Article Title: Dual Processing of FAT1 Cadherin Protein by Human Melanoma Cells Generates Distinct Protein Products *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.234419

    Furin is necessary for production of p430/p85 FAT1 heterodimer, whereas p65 is a cleavage product derived from unprocessed p500 molecule. A , furin-deficient LoVo cells were biotinylated on the cell surface and subjected to immunoprecipitation ( I.P. ) using
    Figure Legend Snippet: Furin is necessary for production of p430/p85 FAT1 heterodimer, whereas p65 is a cleavage product derived from unprocessed p500 molecule. A , furin-deficient LoVo cells were biotinylated on the cell surface and subjected to immunoprecipitation ( I.P. ) using

    Techniques Used: Derivative Assay, Immunoprecipitation

    18) Product Images from "Glycoprotein D actively induces rapid internalization of two nectin-1 isoforms during herpes simplex virus entry"

    Article Title: Glycoprotein D actively induces rapid internalization of two nectin-1 isoforms during herpes simplex virus entry

    Journal: Virology

    doi: 10.1016/j.virol.2009.12.034

    Nectin-1 internalization is triggered by gD. (A) Steps of nectin-1 internalization assay. (B) NGC12 cells were biotinylated and co-cultured with B78 (left panel) or B78-gD(wt) (middle panel) cells for various times before treatment with Proteinase K.
    Figure Legend Snippet: Nectin-1 internalization is triggered by gD. (A) Steps of nectin-1 internalization assay. (B) NGC12 cells were biotinylated and co-cultured with B78 (left panel) or B78-gD(wt) (middle panel) cells for various times before treatment with Proteinase K.

    Techniques Used: Cell Culture

    19) Product Images from "Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors"

    Article Title: Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors

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

    doi: 10.1073/pnas.0407396101

    Detection of oxidized PTEN by biotinylation in cells treated with exogenous H 2 O 2 or with growth factors. NIH 3T3 cells were incubated either for the indicated times with 1 mM H 2 O 2 ( A ) or for 5 min with the indicated concentrations of H 2 O 2 ( B ); HeLa cells were incubated for the indicated times with 100 ng/ml EGF ( C ); NIH 3T3 cells were incubated for the indicated times with 25 ng/ml PDGF ( D ); and HEK293 cells were incubated for the indicated times with 0.5 μg/ml insulin ( E ). Cell lysates were then prepared and subjected to labeling with biotin-conjugated maleimide as described in Materials and Methods . A portion (10%) of each biotinylated sample was subjected to immunoblot analysis with rabbit Abs to PTEN as a control ( Lower ), whereas biotinylated proteins in the remaining fraction were precipitated with avidin-conjugated agarose and then subjected to immunoblot analysis with Abs to PTEN to detect biotinylated PTEN (BM-PTEN) ( Upper ).
    Figure Legend Snippet: Detection of oxidized PTEN by biotinylation in cells treated with exogenous H 2 O 2 or with growth factors. NIH 3T3 cells were incubated either for the indicated times with 1 mM H 2 O 2 ( A ) or for 5 min with the indicated concentrations of H 2 O 2 ( B ); HeLa cells were incubated for the indicated times with 100 ng/ml EGF ( C ); NIH 3T3 cells were incubated for the indicated times with 25 ng/ml PDGF ( D ); and HEK293 cells were incubated for the indicated times with 0.5 μg/ml insulin ( E ). Cell lysates were then prepared and subjected to labeling with biotin-conjugated maleimide as described in Materials and Methods . A portion (10%) of each biotinylated sample was subjected to immunoblot analysis with rabbit Abs to PTEN as a control ( Lower ), whereas biotinylated proteins in the remaining fraction were precipitated with avidin-conjugated agarose and then subjected to immunoblot analysis with Abs to PTEN to detect biotinylated PTEN (BM-PTEN) ( Upper ).

    Techniques Used: Incubation, Labeling, Avidin-Biotin Assay

    20) Product Images from "Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity"

    Article Title: Increased expression of LDL receptor-related protein 1 during human cytomegalovirus infection reduces virion cholesterol and infectivity

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2012.05.012

    Identification of cell surface protein alterations in HCMV-infected cells Fibroblasts were mock-infected or infected at a multiplicity of 5 IU/cell. (A) Specificity of biotin labeling: Avidin-purified cell surface protein preparations were assayed by Western blot for β-actin, insulin receptor (IR), MHC-class I receptor (HLA-B/C), and prostaglandin receptor 2 (PGE2R2). (B) Purification of biotinylated proteins: Avidin-purified samples (~50 µg protein in +biotin samples) were subjected to electrophoresis and Coomassie stained. (C) Reproducibility of MS data across independent experiments: Normalized spectral counts of infected cell proteins isolated at 6 (■), 24 (●) and 72 hpi (▲) were plotted from two independent experiments. Dotted-lines mark a 2-fold difference in counts.
    Figure Legend Snippet: Identification of cell surface protein alterations in HCMV-infected cells Fibroblasts were mock-infected or infected at a multiplicity of 5 IU/cell. (A) Specificity of biotin labeling: Avidin-purified cell surface protein preparations were assayed by Western blot for β-actin, insulin receptor (IR), MHC-class I receptor (HLA-B/C), and prostaglandin receptor 2 (PGE2R2). (B) Purification of biotinylated proteins: Avidin-purified samples (~50 µg protein in +biotin samples) were subjected to electrophoresis and Coomassie stained. (C) Reproducibility of MS data across independent experiments: Normalized spectral counts of infected cell proteins isolated at 6 (■), 24 (●) and 72 hpi (▲) were plotted from two independent experiments. Dotted-lines mark a 2-fold difference in counts.

    Techniques Used: Infection, Labeling, Avidin-Biotin Assay, Purification, Western Blot, Electrophoresis, Staining, Mass Spectrometry, Isolation

    21) Product Images from "Distinct mechanisms survey the structural integrity of HLA-B*27:05 intracellularly and at the surface"

    Article Title: Distinct mechanisms survey the structural integrity of HLA-B*27:05 intracellularly and at the surface

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0200811

    B*27:05-Y84C is more resistant to thermal denaturation and structurally more ordered in the peptide-receptive state. (A) Thermal stability assay. STF1 (upper panel) and STF1-TAP2 cells (lower panel) expressing HA-tagged wild type B*27:05 and B*27:05-Y84C were pulse-labeled with 35 S-methionine/-cysteine for 7 min and then lysed. Detergent lysates were incubated at the temperatures indicated for 10 min prior to sequential immunoprecipitation with W6/32 and anti-HA, respectively. (B) Quantification of thermal stability. Band intensities from lanes representing 4 and 35°C were calculated for the individual samples. Thermal stability of the constructs is calculated and displayed in a bar chart as the ratio of the 35°C signal over the 4°C signal from W6/32 immunoprecipitation (SEM, n = 3). Wild type B*27:05 shows a decrease in thermal stability in the absence of TAP2 (left) whereas B*27:05-Y84C is equally stable in STF1 and STF1-TAP2 cells. (C) β 2 m affinity measurement. Human β 2 m was immunoprecipitated from cell lysates using BBM.1 antibody. Co-immunoprecipitated B*27:05 heavy chains were identified by western blotting using HA antibody (upper row). Where indicated, a high-affinity ligand for B*27:05 (IRAAPPPLF) was added to the lysate prior to immunoprecipitation. STF1-TAP2 cells were used as a positive control for BBM.1 immunoprecipitation. Last lane (CTRL) indicates STF1 cells that do not express B*27:05. Equal amount of lysate (2% of total) was loaded on a separate gel as a loading control (lower row). B*27:05 heavy chains were detected using anti-HA in immunoblotting (IB). (D) Peptide binding to B*27:05. Biotinylated IRAAPPPKF peptide was added to the cells (where indicated) at the point of lysis, and complexed proteins were isolated using streptavidin-conjugated agarose beads. Associated B*27:05 heavy chains were detected using anti-HA antibody in immunoblotting. Last two lanes are loading controls from the lysates (2% of total).
    Figure Legend Snippet: B*27:05-Y84C is more resistant to thermal denaturation and structurally more ordered in the peptide-receptive state. (A) Thermal stability assay. STF1 (upper panel) and STF1-TAP2 cells (lower panel) expressing HA-tagged wild type B*27:05 and B*27:05-Y84C were pulse-labeled with 35 S-methionine/-cysteine for 7 min and then lysed. Detergent lysates were incubated at the temperatures indicated for 10 min prior to sequential immunoprecipitation with W6/32 and anti-HA, respectively. (B) Quantification of thermal stability. Band intensities from lanes representing 4 and 35°C were calculated for the individual samples. Thermal stability of the constructs is calculated and displayed in a bar chart as the ratio of the 35°C signal over the 4°C signal from W6/32 immunoprecipitation (SEM, n = 3). Wild type B*27:05 shows a decrease in thermal stability in the absence of TAP2 (left) whereas B*27:05-Y84C is equally stable in STF1 and STF1-TAP2 cells. (C) β 2 m affinity measurement. Human β 2 m was immunoprecipitated from cell lysates using BBM.1 antibody. Co-immunoprecipitated B*27:05 heavy chains were identified by western blotting using HA antibody (upper row). Where indicated, a high-affinity ligand for B*27:05 (IRAAPPPLF) was added to the lysate prior to immunoprecipitation. STF1-TAP2 cells were used as a positive control for BBM.1 immunoprecipitation. Last lane (CTRL) indicates STF1 cells that do not express B*27:05. Equal amount of lysate (2% of total) was loaded on a separate gel as a loading control (lower row). B*27:05 heavy chains were detected using anti-HA in immunoblotting (IB). (D) Peptide binding to B*27:05. Biotinylated IRAAPPPKF peptide was added to the cells (where indicated) at the point of lysis, and complexed proteins were isolated using streptavidin-conjugated agarose beads. Associated B*27:05 heavy chains were detected using anti-HA antibody in immunoblotting. Last two lanes are loading controls from the lysates (2% of total).

    Techniques Used: Stability Assay, Expressing, Labeling, Incubation, Immunoprecipitation, Construct, Western Blot, Positive Control, Binding Assay, Lysis, Isolation

    22) Product Images from "Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane"

    Article Title: Development of a Proximity Labeling System to Map the Chlamydia trachomatis Inclusion Membrane

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00040

    Lysis conditions compatible with solubilization of inclusion membrane proteins and pulldown of biotinylated proteins . HeLa cells seeded in 6-well plates were infected with Ctr L2 transformant IncF-APEX2, wild-type Ctr L2, or mock infected with the aTc induction/treatment conditions, followed by the biotin-phenol additions as indicated. The cells remaining in the 6-well plates were lysed as described in the Materials and Methods, and cleared lysates (lysates) and the insoluble pellets (pellets) were resolved by SDS-PAGE, transferred to a PVDF membrane, and Western blotted for a transmembrane Golgi protein GM130 (A) , chlamydial heat shock protein 60 (cHsp60) (B) , inclusion membrane protein IncA (C) , eukaryotic cytoskeletal protein tubulin (D) , and biotinylated proteins with streptavidin (E) . Cleared lysates were normalized for protein content and equal amounts of protein were added to magnetic beads conjugated to streptavidin to pull down biotinylated proteins from lysates. Western blot analysis using a streptavidin conjugate of eluate and unbound fractions from the pulldowns are shown in (F) and Coomassie stain of the PVDF membrane showing total protein in (G) .
    Figure Legend Snippet: Lysis conditions compatible with solubilization of inclusion membrane proteins and pulldown of biotinylated proteins . HeLa cells seeded in 6-well plates were infected with Ctr L2 transformant IncF-APEX2, wild-type Ctr L2, or mock infected with the aTc induction/treatment conditions, followed by the biotin-phenol additions as indicated. The cells remaining in the 6-well plates were lysed as described in the Materials and Methods, and cleared lysates (lysates) and the insoluble pellets (pellets) were resolved by SDS-PAGE, transferred to a PVDF membrane, and Western blotted for a transmembrane Golgi protein GM130 (A) , chlamydial heat shock protein 60 (cHsp60) (B) , inclusion membrane protein IncA (C) , eukaryotic cytoskeletal protein tubulin (D) , and biotinylated proteins with streptavidin (E) . Cleared lysates were normalized for protein content and equal amounts of protein were added to magnetic beads conjugated to streptavidin to pull down biotinylated proteins from lysates. Western blot analysis using a streptavidin conjugate of eluate and unbound fractions from the pulldowns are shown in (F) and Coomassie stain of the PVDF membrane showing total protein in (G) .

    Techniques Used: Lysis, Infection, SDS Page, Western Blot, Magnetic Beads, Staining

    23) Product Images from "The Toxoplasma gondii Cyst Wall Interactome"

    Article Title: The Toxoplasma gondii Cyst Wall Interactome

    Journal: mBio

    doi: 10.1128/mBio.02699-19

    Initial cyst wall pulldowns with BioID reveal hypothetical proteins. (A) Schematic diagram of the exogenous expression construct containing the promoter and coding sequence of the cyst wall gene of interest fused C terminally with BirA* and 3×HA followed by the DHFR selectable marker. (B) Immunofluorescence micrographs of Pru and BirA*-tagged parasites (CST1, MAG1, MCP4, BPK1, and GRA6) stained with anti-HA antibody and Alexa Fluor 488-conjugated streptavidin under pH 8 conditions supplemented with 150 μM biotin. Endogenously biotinylated proteins within the apicoplast are observed within these parasite strains. (C) Network graph showing potential parasite-specific interacting proteins of each BirA*-tagged cyst wall protein (CST1, MAG1, MCP4, BPK1, and GRA6) identified during the bradyzoite stage. BirA* cyst wall bait proteins, known cyst wall proteins, dense granule proteins, and hypothetical proteins are represented by purple, red, green, and blue nodes, respectively. Darker arrows correspond to higher normalized spectral abundance factor (NSAF) values of prey proteins identified in each pulldown.
    Figure Legend Snippet: Initial cyst wall pulldowns with BioID reveal hypothetical proteins. (A) Schematic diagram of the exogenous expression construct containing the promoter and coding sequence of the cyst wall gene of interest fused C terminally with BirA* and 3×HA followed by the DHFR selectable marker. (B) Immunofluorescence micrographs of Pru and BirA*-tagged parasites (CST1, MAG1, MCP4, BPK1, and GRA6) stained with anti-HA antibody and Alexa Fluor 488-conjugated streptavidin under pH 8 conditions supplemented with 150 μM biotin. Endogenously biotinylated proteins within the apicoplast are observed within these parasite strains. (C) Network graph showing potential parasite-specific interacting proteins of each BirA*-tagged cyst wall protein (CST1, MAG1, MCP4, BPK1, and GRA6) identified during the bradyzoite stage. BirA* cyst wall bait proteins, known cyst wall proteins, dense granule proteins, and hypothetical proteins are represented by purple, red, green, and blue nodes, respectively. Darker arrows correspond to higher normalized spectral abundance factor (NSAF) values of prey proteins identified in each pulldown.

    Techniques Used: Expressing, Construct, Sequencing, Marker, Immunofluorescence, Staining

    24) Product Images from "Statins stimulate the production of a soluble form of the receptor for advanced glycation end products"

    Article Title: Statins stimulate the production of a soluble form of the receptor for advanced glycation end products

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M038968

    Impact of lovastatin treatment on full-length RAGE and ADAM10 localization on the cell surface. HEK/RAGE cells cultured in lipid-deficient medium for 24 h in the presence of 2 μM lovastatin or equivalent amounts of water (control), were biotinylated
    Figure Legend Snippet: Impact of lovastatin treatment on full-length RAGE and ADAM10 localization on the cell surface. HEK/RAGE cells cultured in lipid-deficient medium for 24 h in the presence of 2 μM lovastatin or equivalent amounts of water (control), were biotinylated

    Techniques Used: Cell Culture

    25) Product Images from "Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication ▿"

    Article Title: Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02039-09

    Localization of LASV proteins in detergent-soluble membrane areas. (A) Flotation analysis of transiently LASV GP-expressing cells. Cells expressing LASV GP, VSV G, or influenza virus HA, respectively, were solubilized with 0.5% Triton X-100 at 4°C, and the lysate was subjected to flotation in a sucrose gradient. Gradient fractions were collected from the top after ultracentrifugation and analyzed using SDS-PAGE and Western blotting with the antiserum anti-GP-2-C, anti-HA, or anti-VSV G. The influenza virus HA, a DRM-located protein, and VSV glycoprotein G, a DRM-excluded protein, served as controls. (B) Cell surface distribution of LASV GP after Triton X-100 treatment. Transiently LASV GP-expressing cells were cell surface labeled with biotin. Then, cells were lysed in TNE buffer containing 0.5% Triton X-100 at 4°C and separated into detergent-soluble (s) and detergent-insoluble (i) fractions by centrifugation. Biotinylated proteins from each fraction were precipitated using streptavidin-coupled beads and subjected to SDS-PAGE and immunoblotting. LASV GP was stained with an anti-GP-2-C antibody. As a marker for detergent-insoluble fractions, GM1 was visualized on a nitrocellulose membrane by incubation with HRP-coupled cholera toxin subunit B. The number symbol denotes unspecific protein bands. (C) Effect of Triton X-100 treatment on LASV GP in infected cells. Cells were infected with LASV at an MOI of 1. Two days postinfection, they were divided into detergent-soluble (s) and detergent-insoluble (i) fractions and analyzed as described for panel B. Immunoblots were stained with an anti-GP-2-C, anti-NP, or anti-Z antibody. GM1 was visualized as described for panel B. The number symbol denotes unspecific protein bands.
    Figure Legend Snippet: Localization of LASV proteins in detergent-soluble membrane areas. (A) Flotation analysis of transiently LASV GP-expressing cells. Cells expressing LASV GP, VSV G, or influenza virus HA, respectively, were solubilized with 0.5% Triton X-100 at 4°C, and the lysate was subjected to flotation in a sucrose gradient. Gradient fractions were collected from the top after ultracentrifugation and analyzed using SDS-PAGE and Western blotting with the antiserum anti-GP-2-C, anti-HA, or anti-VSV G. The influenza virus HA, a DRM-located protein, and VSV glycoprotein G, a DRM-excluded protein, served as controls. (B) Cell surface distribution of LASV GP after Triton X-100 treatment. Transiently LASV GP-expressing cells were cell surface labeled with biotin. Then, cells were lysed in TNE buffer containing 0.5% Triton X-100 at 4°C and separated into detergent-soluble (s) and detergent-insoluble (i) fractions by centrifugation. Biotinylated proteins from each fraction were precipitated using streptavidin-coupled beads and subjected to SDS-PAGE and immunoblotting. LASV GP was stained with an anti-GP-2-C antibody. As a marker for detergent-insoluble fractions, GM1 was visualized on a nitrocellulose membrane by incubation with HRP-coupled cholera toxin subunit B. The number symbol denotes unspecific protein bands. (C) Effect of Triton X-100 treatment on LASV GP in infected cells. Cells were infected with LASV at an MOI of 1. Two days postinfection, they were divided into detergent-soluble (s) and detergent-insoluble (i) fractions and analyzed as described for panel B. Immunoblots were stained with an anti-GP-2-C, anti-NP, or anti-Z antibody. GM1 was visualized as described for panel B. The number symbol denotes unspecific protein bands.

    Techniques Used: Expressing, SDS Page, Western Blot, Labeling, Centrifugation, Staining, Marker, Incubation, Infection

    Oligomeric state of LASV glycoprotein expressed in mammalian cells. (A) LASV glycoprotein present in cells analyzed by sucrose gradient ultracentrifugation. LASV glycoprotein-expressing cells were lysed with 1% Triton X-100, and cell lysate was subjected to sucrose gradient ultracentrifugation. Aliquots of each gradient fraction were subjected to SDS-PAGE and immunoblotting using the antibody anti-GP-1 (panel 1) or an anti-GP-2-C antiserum (panel 2). GP-C, GP-1, and GP-2 on the immunoblots were quantified (panels 3 to 5). The positions of the gradient marker proteins bovine serum albumin (67 kDa), catalase (232 kDa), and thyroglobulin (669 kDa) are indicated. (B) Comparison of oligomeric forms of GP-C on the cell surface with total GP-C. Cell surface proteins were biotinylated before cell lysis. Lysates of biotinylated and nonbiotinylated cells were subjected to sucrose gradient ultracentrifugation. Biotinylated GP-C was precipitated from gradient fractions using streptavidin-coupled Sepharose beads. Both protein samples, the biotinylated sample (broken line) and the nonbiotinylated control (solid line), were subjected to SDS-PAGE and immunoblotting using anti-GP-2-C antiserum.
    Figure Legend Snippet: Oligomeric state of LASV glycoprotein expressed in mammalian cells. (A) LASV glycoprotein present in cells analyzed by sucrose gradient ultracentrifugation. LASV glycoprotein-expressing cells were lysed with 1% Triton X-100, and cell lysate was subjected to sucrose gradient ultracentrifugation. Aliquots of each gradient fraction were subjected to SDS-PAGE and immunoblotting using the antibody anti-GP-1 (panel 1) or an anti-GP-2-C antiserum (panel 2). GP-C, GP-1, and GP-2 on the immunoblots were quantified (panels 3 to 5). The positions of the gradient marker proteins bovine serum albumin (67 kDa), catalase (232 kDa), and thyroglobulin (669 kDa) are indicated. (B) Comparison of oligomeric forms of GP-C on the cell surface with total GP-C. Cell surface proteins were biotinylated before cell lysis. Lysates of biotinylated and nonbiotinylated cells were subjected to sucrose gradient ultracentrifugation. Biotinylated GP-C was precipitated from gradient fractions using streptavidin-coupled Sepharose beads. Both protein samples, the biotinylated sample (broken line) and the nonbiotinylated control (solid line), were subjected to SDS-PAGE and immunoblotting using anti-GP-2-C antiserum.

    Techniques Used: Expressing, SDS Page, Western Blot, Marker, Lysis

    26) Product Images from "Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells"

    Article Title: Distinct domain-dependent effect of syntaxin1A on amiloride-sensitive sodium channel (ENaC) currents in HT-29 colonic epithelial cells

    Journal: International Journal of Biological Sciences

    doi:

    Syntaxin1A domains modulate ENaC expression at the cell surface - HT-29 cells were transfected with wild-type syntaxin1A and its truncated constructs. Cell surface proteins were biotinylated with Sulpho-NHS-SS-biotin, pulled down with streptavidin-agarose separated by SDS-PAGE and transferred to PVDF membrane. The blots were probed with γENaC antibody (A). The protein was analyzed by densitometry (B). The data reflect increased expression of ENaC in HT-29 cells transfected with syntaxin1A, which is reflected in enhanced amiloride-sensitive currents reported in the text. Data represent three individual experiments each performed with different batches of HT-29 cells.
    Figure Legend Snippet: Syntaxin1A domains modulate ENaC expression at the cell surface - HT-29 cells were transfected with wild-type syntaxin1A and its truncated constructs. Cell surface proteins were biotinylated with Sulpho-NHS-SS-biotin, pulled down with streptavidin-agarose separated by SDS-PAGE and transferred to PVDF membrane. The blots were probed with γENaC antibody (A). The protein was analyzed by densitometry (B). The data reflect increased expression of ENaC in HT-29 cells transfected with syntaxin1A, which is reflected in enhanced amiloride-sensitive currents reported in the text. Data represent three individual experiments each performed with different batches of HT-29 cells.

    Techniques Used: Expressing, Transfection, Construct, SDS Page

    27) Product Images from "Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration *"

    Article Title: Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.427765

    The majority of H-Ras is not palmitoylated. A , hydroxylamine (HA)-dependent biotin switch is a reversed labeling technique for palmitoylated cysteines. First, free thiols (RSH) are alkylated by maleimide. Then thioester bonds, including palmitoylation, are hydrolyzed by HA, whereas reversible oxidative ( ox ) modifications are removed by DTT. The freed cysteines are subsequently labeled by thiol-reactive HPDP-Biotin ( B-HPDP ), and biotinylated protein is enriched using streptavidin pull-down. B , palmitoylation of endogenous H-Ras was measured using the HA biotin switch assay. Lysates were treated either with DTT and HA to label total H-Ras or with HA only to specifically remove S -palmitoylation and label freed cysteines with thiol-reactive HPDP-Biotin. The pull-down on streptavidin-agarose ( Strep ) was separated by SDS-PAGE and immunoblotted for H-Ras with an isoform-specific antibody. IB , immunoblot; IP , immunoprecipitation. C , densitometry was performed for streptavidin pull-down ( n = 3). Error bars , S.E.
    Figure Legend Snippet: The majority of H-Ras is not palmitoylated. A , hydroxylamine (HA)-dependent biotin switch is a reversed labeling technique for palmitoylated cysteines. First, free thiols (RSH) are alkylated by maleimide. Then thioester bonds, including palmitoylation, are hydrolyzed by HA, whereas reversible oxidative ( ox ) modifications are removed by DTT. The freed cysteines are subsequently labeled by thiol-reactive HPDP-Biotin ( B-HPDP ), and biotinylated protein is enriched using streptavidin pull-down. B , palmitoylation of endogenous H-Ras was measured using the HA biotin switch assay. Lysates were treated either with DTT and HA to label total H-Ras or with HA only to specifically remove S -palmitoylation and label freed cysteines with thiol-reactive HPDP-Biotin. The pull-down on streptavidin-agarose ( Strep ) was separated by SDS-PAGE and immunoblotted for H-Ras with an isoform-specific antibody. IB , immunoblot; IP , immunoprecipitation. C , densitometry was performed for streptavidin pull-down ( n = 3). Error bars , S.E.

    Techniques Used: Labeling, Biotin Switch Assay, SDS Page, Immunoprecipitation

    28) Product Images from "Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment *"

    Article Title: Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.603977

    SIRT2 overexpression acts through the TUG C terminus to reduce intracellular retention of GSV cargos. A , cell surface-exposed IRAP was biotinylated in basal and insulin-stimulated 3T3-L1 adipocytes. Control and SIRT2-overexpressing cells containing WT
    Figure Legend Snippet: SIRT2 overexpression acts through the TUG C terminus to reduce intracellular retention of GSV cargos. A , cell surface-exposed IRAP was biotinylated in basal and insulin-stimulated 3T3-L1 adipocytes. Control and SIRT2-overexpressing cells containing WT

    Techniques Used: Over Expression

    29) Product Images from "Phosphorylation of MUC1 by Met Modulates Interaction with p53 and MMP1 Expression *Phosphorylation of MUC1 by Met Modulates Interaction with p53 and MMP1 Expression * S⃞"

    Article Title: Phosphorylation of MUC1 by Met Modulates Interaction with p53 and MMP1 Expression *Phosphorylation of MUC1 by Met Modulates Interaction with p53 and MMP1 Expression * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M805036200

    MUC1 promotes endocytosis, lysosomal degradation, and reduced signaling of Met. A, effect of MUC1 overexpression on Met endocytosis was determined in S2-013.MUC1F or S2-013.neo cells. Serum-starved cells were surface-biotinylated on ice and then incubated at 37 °C for 2 h in the absence (serum-free or SF ) or presence of HGF ( H ) to allow endocytosis. Surface-biotinylated proteins from RIPA lysates were recovered on neutravidin beads and analyzed by SDS-PAGE, followed by Western blotting with a polyclonal Ab against Met. 1st 3 lanes indicate the total levels of surface-biotinylated Met after 2 h of incubation at 0, 37, or 37 °C in the presence of HGF and ammonium chloride, and 4th to 6th lanes represent the levels of endocytosed Met (detected by glutathione stripping or GS ) under serum-free conditions or HGF stimulation with and without ammonium chloride. B, effect of HGF stimulation on Met signaling. S2-013.MUC1F or S2-013.neo cells were stimulated with HGF for different times. Lysates were immunoprecipitated with Met cytoplasmic tail Ab or IgG control. The immunoprecipitants were resolved on SDS-PAGE and immunoblotted ( IB ) with Abs against Gab1 or Met.
    Figure Legend Snippet: MUC1 promotes endocytosis, lysosomal degradation, and reduced signaling of Met. A, effect of MUC1 overexpression on Met endocytosis was determined in S2-013.MUC1F or S2-013.neo cells. Serum-starved cells were surface-biotinylated on ice and then incubated at 37 °C for 2 h in the absence (serum-free or SF ) or presence of HGF ( H ) to allow endocytosis. Surface-biotinylated proteins from RIPA lysates were recovered on neutravidin beads and analyzed by SDS-PAGE, followed by Western blotting with a polyclonal Ab against Met. 1st 3 lanes indicate the total levels of surface-biotinylated Met after 2 h of incubation at 0, 37, or 37 °C in the presence of HGF and ammonium chloride, and 4th to 6th lanes represent the levels of endocytosed Met (detected by glutathione stripping or GS ) under serum-free conditions or HGF stimulation with and without ammonium chloride. B, effect of HGF stimulation on Met signaling. S2-013.MUC1F or S2-013.neo cells were stimulated with HGF for different times. Lysates were immunoprecipitated with Met cytoplasmic tail Ab or IgG control. The immunoprecipitants were resolved on SDS-PAGE and immunoblotted ( IB ) with Abs against Gab1 or Met.

    Techniques Used: Over Expression, Incubation, SDS Page, Western Blot, Stripping Membranes, Immunoprecipitation

    30) Product Images from "A Surface-Focused Biotinylation Procedure Identifies the Yersinia pestis Catalase KatY as a Membrane-Associated but Non-Surface-Located Protein ▿"

    Article Title: A Surface-Focused Biotinylation Procedure Identifies the Yersinia pestis Catalase KatY as a Membrane-Associated but Non-Surface-Located Protein ▿

    Journal:

    doi: 10.1128/AEM.02968-06

    Mass spectrometric analysis of surface-biotinylated proteins.
    Figure Legend Snippet: Mass spectrometric analysis of surface-biotinylated proteins.

    Techniques Used:

    31) Product Images from "Disruption of Cdk5-Associated Phosphorylation of Residue Threonine-161 of the δ-Opioid Receptor: Impaired Receptor Function and Attenuated Morphine Antinociceptive Tolerance"

    Article Title: Disruption of Cdk5-Associated Phosphorylation of Residue Threonine-161 of the δ-Opioid Receptor: Impaired Receptor Function and Attenuated Morphine Antinociceptive Tolerance

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0415-09.2009

    Thr-161 is required for cell surface expression of DOR and the formation of DOR–MOR heterodimers. A , No significant changes in the total expression level between wild-type DOR (WT) and T161A-DOR after transient transfection in NG108-15 cells. B , Surface-biotinylated DOR showed that the expression of functional DOR at the cell surface for the T161A mutant was significantly less than that of WT-DOR after transient transfection in NG108-15 cells. C , Cell lysates from NG108-15 cells cotransfected with EGFP-DOR (2 μg), HA-MOR (4 μg) cDNA, and Cdk5 siRNA or control siRNA were subjected to immunoprecipitation using polyclonal anti-GFP antibody. Associated HA-MOR was detected by immunoblotting using a monoclonal anti-HA antibody. The levels of DOR and MOR in each transfection are shown by immunoblotting with anti-GFP and anti-HA antibodies, respectively. D , Cell lysates from NG108-15 cells cotransfected with EGFP-DOR (wild type or T161A) (2 μg) and HA-MOR (4 μg) cDNA were subjected to immunoprecipitation using polyclonal anti-GFP antibody. Associated HA-MOR was detected by immunoblotting using a monoclonal anti-HA antibody. The levels of DOR and MOR in each transfection are shown by immunoblotting with anti-GFP and anti-HA antibodies, respectively. Results are representative of three independent experiments.
    Figure Legend Snippet: Thr-161 is required for cell surface expression of DOR and the formation of DOR–MOR heterodimers. A , No significant changes in the total expression level between wild-type DOR (WT) and T161A-DOR after transient transfection in NG108-15 cells. B , Surface-biotinylated DOR showed that the expression of functional DOR at the cell surface for the T161A mutant was significantly less than that of WT-DOR after transient transfection in NG108-15 cells. C , Cell lysates from NG108-15 cells cotransfected with EGFP-DOR (2 μg), HA-MOR (4 μg) cDNA, and Cdk5 siRNA or control siRNA were subjected to immunoprecipitation using polyclonal anti-GFP antibody. Associated HA-MOR was detected by immunoblotting using a monoclonal anti-HA antibody. The levels of DOR and MOR in each transfection are shown by immunoblotting with anti-GFP and anti-HA antibodies, respectively. D , Cell lysates from NG108-15 cells cotransfected with EGFP-DOR (wild type or T161A) (2 μg) and HA-MOR (4 μg) cDNA were subjected to immunoprecipitation using polyclonal anti-GFP antibody. Associated HA-MOR was detected by immunoblotting using a monoclonal anti-HA antibody. The levels of DOR and MOR in each transfection are shown by immunoblotting with anti-GFP and anti-HA antibodies, respectively. Results are representative of three independent experiments.

    Techniques Used: Expressing, Transfection, Functional Assay, Mutagenesis, Immunoprecipitation

    32) Product Images from "Aquaporin-4 Cell-Surface Expression and Turnover Are Regulated by Dystroglycan, Dynamin, and the Extracellular Matrix in Astrocytes"

    Article Title: Aquaporin-4 Cell-Surface Expression and Turnover Are Regulated by Dystroglycan, Dynamin, and the Extracellular Matrix in Astrocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165439

    Dystroglycan and laminin are involved in the regulation of AQP4 cell-surface expression in astrocytes. (A) The cell-surface, intracellular, and total (Input) fractions from control untreated astrocytes ( - LN) and astrocytes treated with 24 nM laminin-111 (+LN) were immunoblotted for AQP4 and β-DG. (B) Histogram summarizing the differences in protein expression levels of AQP4 at the cell surface of untreated and laminin-treated astrocytes, normalized against β-DG levels. Values represent relative normalized mean pixel intensities ±SEM from three different experiments. The asterisk indicates a statistically significant increase of AQP4 expression compared to untreated astrocytes, as determined by a two-tailed Student’s t -test (*p = 0.033). (C) Cell-surface proteins isolated via biotinylation assay from siCTL and siDAG1-transfected astrocytes were immunoblotted for AQP4, β-DG, and with streptavidin-HRP to visualize all biotinylated proteins. (D) Quantification of cell-surface AQP4 levels, normalized against all biotinylated species, visualized using streptavidin-HRP, for three independent experiments. Asterisk indicates statistical significance, as determined by Students's t -test (*p = 0.020).
    Figure Legend Snippet: Dystroglycan and laminin are involved in the regulation of AQP4 cell-surface expression in astrocytes. (A) The cell-surface, intracellular, and total (Input) fractions from control untreated astrocytes ( - LN) and astrocytes treated with 24 nM laminin-111 (+LN) were immunoblotted for AQP4 and β-DG. (B) Histogram summarizing the differences in protein expression levels of AQP4 at the cell surface of untreated and laminin-treated astrocytes, normalized against β-DG levels. Values represent relative normalized mean pixel intensities ±SEM from three different experiments. The asterisk indicates a statistically significant increase of AQP4 expression compared to untreated astrocytes, as determined by a two-tailed Student’s t -test (*p = 0.033). (C) Cell-surface proteins isolated via biotinylation assay from siCTL and siDAG1-transfected astrocytes were immunoblotted for AQP4, β-DG, and with streptavidin-HRP to visualize all biotinylated proteins. (D) Quantification of cell-surface AQP4 levels, normalized against all biotinylated species, visualized using streptavidin-HRP, for three independent experiments. Asterisk indicates statistical significance, as determined by Students's t -test (*p = 0.020).

    Techniques Used: Expressing, Two Tailed Test, Isolation, Cell Surface Biotinylation Assay, Transfection

    Laminin affects the cell-surface expression of only the M23 isoform of AQP4. (A) Laminin-treated (+LN) and untreated control CHO cells expressing GFP-tagged M1 and M23 isoforms of AQP4 were subjected to biotinylation analysis, and the input and biotinylated (Cell surface) fractions were isolated and probed for the proteins indicated. (B) Histogram summarizes the results obtained over 4 such experiments. The asterisk indicates a statistically-significant difference, as determined via the Students t -test (*p = 0.0408).
    Figure Legend Snippet: Laminin affects the cell-surface expression of only the M23 isoform of AQP4. (A) Laminin-treated (+LN) and untreated control CHO cells expressing GFP-tagged M1 and M23 isoforms of AQP4 were subjected to biotinylation analysis, and the input and biotinylated (Cell surface) fractions were isolated and probed for the proteins indicated. (B) Histogram summarizes the results obtained over 4 such experiments. The asterisk indicates a statistically-significant difference, as determined via the Students t -test (*p = 0.0408).

    Techniques Used: Expressing, Isolation

    33) Product Images from "High Fat Diet Enhances β-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting β-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation"

    Article Title: High Fat Diet Enhances β-Site Cleavage of Amyloid Precursor Protein (APP) via Promoting β-Site APP Cleaving Enzyme 1/Adaptor Protein 2/Clathrin Complex Formation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0131199

    Overexpression of AP-2 promoted the cleavage of APP by BACE1 in Swedish APP overexpressing CHO cells and in SH-SY5Y cells. (A) APPsw/Ncad-CHO cells were transfected either with WT BACE1 and empty vector (1 st and 3 rd lanes) or with WT BACE1 and AP-2α (2 nd and 4 th lanes). Myc tag was linked to WT BACE1 and HA tag was linked to AP-2α. Equal amounts of cell lysate were immunoprecipitated by an anti-clathrin antibody, and then immunoblotted by an anti-myc antibody (upper panel, 1 st and 2 nd lanes). As a negative control, samples were immunoprecipitated by a normal mouse IgG (3 rd and 4 th lanes). The CHO cells overexpressing either with WT BACE1 and empty vector or with WT BACE1 and AP-2α were biotinylated, and cell surface level of BACE1 was examined by an anti-myc antibody (middle panel, 1 st row). As a positive control, cell surface level of TfR was examined (middle panel, 2 nd row). The expression levels of each protein in cell lysates are shown in the bottom panel. Overexpression of AP-2 increased the amount of BACE1/clathrin complex, and decreased the cell surface level of BACE1. (B) Immunostaining analysis to examine the localizations of BACE1 using an anti-myc antibody under AP-2 overexpressing conditions. APPsw/Ncad-CHO cells were transfected with WT BACE1 and AP-2α (right panel). As a negative control, the cells were overexpressing WT BACE1 and empty vector (left panel). Myc tag was linked to WT BACE1. WT BACE1 was localized on the cell surface (arrowhead) as well as in the intracellular compartments in a negative control cells. On the other hand, WT BACE1was hardly localized on the plasma membrane and mainly localized in the intracellular compartments under AP-2 overexpressing conditions. Scale bar indicates 10 μm. (C) Immunostaining analysis to examine the detailed localizations of BACE1 using anti-EEA1 and anti-Lamp2 antibodies under AP-2 overexpressing conditions. Myc tag was linked to WT BACE1. EEA1 was used as an endosome marker and Lamp2 was a lysosome marker. Under the AP-2 overexpressing condition, WT BACE1 was localized in the endosome and in the lysosome. (D) An internalization assay to examine the localization change of cell surface BACE1. APPsw/Ncad-CHO cells were transfected with WT BACE1 and empty vector (left panel) or with WT BACE1 and AP-2α (right panel). The cell surface BACE1 was labeled by an anti-myc antibody, followed by examination of its localization before (upper row) and 15 minutes after incubation (middle row). The expression of AP-2α was checked by an anti-HA antibody (bottom row). 15 minutes after incubation, the BACE1 labelled by an anti-myc antibody was observed on the cell membrane (arrowhead) and at the intracellular vesicles (arrow) in empty vector-transfected cells. On the other hand, the BACE1 was clearly accumulated in the intracellular compartment (arrow) and it was hardly observed on the cell membrane in the AP-2 overexpresing cells. (E) Triple immunostaining analysis for detecting BACE1/AP-2/clathrin complex in the internalization assay. APPsw/Ncad-CHO cells were transfected with WT BACE1 and AP-2α. Cell surface BACE1 was labelled by an anti-myc antibody. Clathrin and AP-2 were stained with anti-clathrin and anti-HA antibodies respectively. BACE1/AP-2/clathrin complex formed during the internalization of cell surface BACE1 (5 minutes after incubation) is indicated arrow. Right upper panel is a merged image and right bottom panel is high magnification of the image contained in the rectangle in the upper panel. Scale bar indicates 10 μm. (F) Immunostaining analysis to examine whether the internalized BACE1 co-localized with APP under AP-2 overexpressing conditions. Myc tag was linked to WT BACE1. The cell surface BACE1 was labelled by an anti-myc antibody, followed by incubating for 15 minutes. APP was stained by 6E10 antibody. 15 minutes after incubation, the BACE1 labelled by an anti-myc antibody was not clearly co-localized with APP. On the other hand, APP remarkably surrounded the compartment in which the BACE1 was accumulated. Scale bar indicates 10 μm. (G) APPsw/Ncad-CHO cells were transfected with WT BACE1 and empty vector (1 st lane), with WT BACE1 and AP-2α (2 nd lane) or with D495R BACE1 and AP-2α (3 rd lane). The levels of extracellular Swedish sAPPβ were determined by the Swedish sAPPβ specific ELISA kit. The levels of sAPPβ were normalized by the levels of BACE1 (mature form plus immature) transiently overexpressed. The average level of sAPPβ in WT BACE1 with empty vector-transfected cells was regarded as 100% and that in other cells was relatively indicated. The level of Swedish sAPPβ in WT BACE1 and AP-2α-transfected cell was significantly higher than that in WT BACE1 and empty vector (n = 12, F (2,33) = 7.86, p = 0.001). On the other hand, the level in D495R BACE1 and AP-2α-transfected cell was lower than that in WT BACE1 and AP-2α-transfected cell (p = 0.002). * indicates p
    Figure Legend Snippet: Overexpression of AP-2 promoted the cleavage of APP by BACE1 in Swedish APP overexpressing CHO cells and in SH-SY5Y cells. (A) APPsw/Ncad-CHO cells were transfected either with WT BACE1 and empty vector (1 st and 3 rd lanes) or with WT BACE1 and AP-2α (2 nd and 4 th lanes). Myc tag was linked to WT BACE1 and HA tag was linked to AP-2α. Equal amounts of cell lysate were immunoprecipitated by an anti-clathrin antibody, and then immunoblotted by an anti-myc antibody (upper panel, 1 st and 2 nd lanes). As a negative control, samples were immunoprecipitated by a normal mouse IgG (3 rd and 4 th lanes). The CHO cells overexpressing either with WT BACE1 and empty vector or with WT BACE1 and AP-2α were biotinylated, and cell surface level of BACE1 was examined by an anti-myc antibody (middle panel, 1 st row). As a positive control, cell surface level of TfR was examined (middle panel, 2 nd row). The expression levels of each protein in cell lysates are shown in the bottom panel. Overexpression of AP-2 increased the amount of BACE1/clathrin complex, and decreased the cell surface level of BACE1. (B) Immunostaining analysis to examine the localizations of BACE1 using an anti-myc antibody under AP-2 overexpressing conditions. APPsw/Ncad-CHO cells were transfected with WT BACE1 and AP-2α (right panel). As a negative control, the cells were overexpressing WT BACE1 and empty vector (left panel). Myc tag was linked to WT BACE1. WT BACE1 was localized on the cell surface (arrowhead) as well as in the intracellular compartments in a negative control cells. On the other hand, WT BACE1was hardly localized on the plasma membrane and mainly localized in the intracellular compartments under AP-2 overexpressing conditions. Scale bar indicates 10 μm. (C) Immunostaining analysis to examine the detailed localizations of BACE1 using anti-EEA1 and anti-Lamp2 antibodies under AP-2 overexpressing conditions. Myc tag was linked to WT BACE1. EEA1 was used as an endosome marker and Lamp2 was a lysosome marker. Under the AP-2 overexpressing condition, WT BACE1 was localized in the endosome and in the lysosome. (D) An internalization assay to examine the localization change of cell surface BACE1. APPsw/Ncad-CHO cells were transfected with WT BACE1 and empty vector (left panel) or with WT BACE1 and AP-2α (right panel). The cell surface BACE1 was labeled by an anti-myc antibody, followed by examination of its localization before (upper row) and 15 minutes after incubation (middle row). The expression of AP-2α was checked by an anti-HA antibody (bottom row). 15 minutes after incubation, the BACE1 labelled by an anti-myc antibody was observed on the cell membrane (arrowhead) and at the intracellular vesicles (arrow) in empty vector-transfected cells. On the other hand, the BACE1 was clearly accumulated in the intracellular compartment (arrow) and it was hardly observed on the cell membrane in the AP-2 overexpresing cells. (E) Triple immunostaining analysis for detecting BACE1/AP-2/clathrin complex in the internalization assay. APPsw/Ncad-CHO cells were transfected with WT BACE1 and AP-2α. Cell surface BACE1 was labelled by an anti-myc antibody. Clathrin and AP-2 were stained with anti-clathrin and anti-HA antibodies respectively. BACE1/AP-2/clathrin complex formed during the internalization of cell surface BACE1 (5 minutes after incubation) is indicated arrow. Right upper panel is a merged image and right bottom panel is high magnification of the image contained in the rectangle in the upper panel. Scale bar indicates 10 μm. (F) Immunostaining analysis to examine whether the internalized BACE1 co-localized with APP under AP-2 overexpressing conditions. Myc tag was linked to WT BACE1. The cell surface BACE1 was labelled by an anti-myc antibody, followed by incubating for 15 minutes. APP was stained by 6E10 antibody. 15 minutes after incubation, the BACE1 labelled by an anti-myc antibody was not clearly co-localized with APP. On the other hand, APP remarkably surrounded the compartment in which the BACE1 was accumulated. Scale bar indicates 10 μm. (G) APPsw/Ncad-CHO cells were transfected with WT BACE1 and empty vector (1 st lane), with WT BACE1 and AP-2α (2 nd lane) or with D495R BACE1 and AP-2α (3 rd lane). The levels of extracellular Swedish sAPPβ were determined by the Swedish sAPPβ specific ELISA kit. The levels of sAPPβ were normalized by the levels of BACE1 (mature form plus immature) transiently overexpressed. The average level of sAPPβ in WT BACE1 with empty vector-transfected cells was regarded as 100% and that in other cells was relatively indicated. The level of Swedish sAPPβ in WT BACE1 and AP-2α-transfected cell was significantly higher than that in WT BACE1 and empty vector (n = 12, F (2,33) = 7.86, p = 0.001). On the other hand, the level in D495R BACE1 and AP-2α-transfected cell was lower than that in WT BACE1 and AP-2α-transfected cell (p = 0.002). * indicates p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control, Positive Control, Expressing, Immunostaining, Marker, Labeling, Incubation, Triple Immunostaining, Staining, Enzyme-linked Immunosorbent Assay

    34) Product Images from "Regulation of Kir4.1 and AQP4 expression and stability at the basolateral domain of epithelial MDCK cells by the extracellular matrix"

    Article Title: Regulation of Kir4.1 and AQP4 expression and stability at the basolateral domain of epithelial MDCK cells by the extracellular matrix

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00315.2010

    FN and LN increase cell surface expression of Kir4.1 and AQP4 at the basolateral domains of MDCK cells. Biotinylated cell surface fractions from control cells (CTL) and cells grown on either FN (5.0 or 50.0 μg/ml) or LN (9.6 or 50.0 μg/ml)
    Figure Legend Snippet: FN and LN increase cell surface expression of Kir4.1 and AQP4 at the basolateral domains of MDCK cells. Biotinylated cell surface fractions from control cells (CTL) and cells grown on either FN (5.0 or 50.0 μg/ml) or LN (9.6 or 50.0 μg/ml)

    Techniques Used: Expressing, CTL Assay

    DG is not the only LN receptor involved in the LN-induced increase in cell surface expression of Kir4.1 and AQP4 in MDCK cells. Biotinylated cell surface fractions from control (siCTL) and Dag1 siRNA-transfected (si Dag1 ) GFP-Kir4.1 and GFP-AQP4-expressing
    Figure Legend Snippet: DG is not the only LN receptor involved in the LN-induced increase in cell surface expression of Kir4.1 and AQP4 in MDCK cells. Biotinylated cell surface fractions from control (siCTL) and Dag1 siRNA-transfected (si Dag1 ) GFP-Kir4.1 and GFP-AQP4-expressing

    Techniques Used: Expressing, Transfection

    Kir4.1 and AQP4 are expressed predominantly within the basolateral membrane domain of MDCK cells. Biotinylated cell surface fractions from the apical (AP) and basolateral (BL) domains of confluent MDCK cells expressing either GFP-Kir4.1 or GFP-AQP4 were
    Figure Legend Snippet: Kir4.1 and AQP4 are expressed predominantly within the basolateral membrane domain of MDCK cells. Biotinylated cell surface fractions from the apical (AP) and basolateral (BL) domains of confluent MDCK cells expressing either GFP-Kir4.1 or GFP-AQP4 were

    Techniques Used: Expressing

    35) Product Images from "A Plasma Membrane Wound Proteome"

    Article Title: A Plasma Membrane Wound Proteome

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.110015

    Retention and redistribution of biotinylated proteins in scraped MEFs. A , MEFs were scraped, labeled with 2 m m MPB, and washed three times with PBS, as described under “Experimental Procedures.” A sample of the first PBS wash was saved
    Figure Legend Snippet: Retention and redistribution of biotinylated proteins in scraped MEFs. A , MEFs were scraped, labeled with 2 m m MPB, and washed three times with PBS, as described under “Experimental Procedures.” A sample of the first PBS wash was saved

    Techniques Used: Labeling

    Wound-associated caldesmon was rapidly degraded by calpain. A , IEC-6 cells and HFL1 fibroblasts were scrape-damaged, and biotinylated wound-associated proteins were isolated as described under “Experimental Procedures” ( lanes marked S
    Figure Legend Snippet: Wound-associated caldesmon was rapidly degraded by calpain. A , IEC-6 cells and HFL1 fibroblasts were scrape-damaged, and biotinylated wound-associated proteins were isolated as described under “Experimental Procedures” ( lanes marked S

    Techniques Used: Isolation

    Isolation of intracellular MEF proteins labeled with MPB after scrape damage. Capns1 -null and wild-type MEFs were scraped and labeled with 0.4 m m MPB using the improved biotinylation method described under “Experimental Procedures.” Biotinylated
    Figure Legend Snippet: Isolation of intracellular MEF proteins labeled with MPB after scrape damage. Capns1 -null and wild-type MEFs were scraped and labeled with 0.4 m m MPB using the improved biotinylation method described under “Experimental Procedures.” Biotinylated

    Techniques Used: Isolation, Labeling

    Lamin C biotinylation after scrape damage did not require vimentin and was transitory. A , vimentin knock-out (−/−) and wild-type (+/+) mouse fibroblast cell lines were subjected to scrape damage and MPB-labeled. Biotinylated proteins were
    Figure Legend Snippet: Lamin C biotinylation after scrape damage did not require vimentin and was transitory. A , vimentin knock-out (−/−) and wild-type (+/+) mouse fibroblast cell lines were subjected to scrape damage and MPB-labeled. Biotinylated proteins were

    Techniques Used: Knock-Out, Labeling

    Intracellular proteins exposed after glass bead crush injury. A , C 2 C 12 myoblasts were injured by crushing with glass beads, as described under “Experimental Procedures.” MPB-biotinylated proteins were isolated and analyzed by SDS-PAGE
    Figure Legend Snippet: Intracellular proteins exposed after glass bead crush injury. A , C 2 C 12 myoblasts were injured by crushing with glass beads, as described under “Experimental Procedures.” MPB-biotinylated proteins were isolated and analyzed by SDS-PAGE

    Techniques Used: Isolation, SDS Page

    36) Product Images from "Fragmentation of Fibronectin by Inherent Autolytic and Matrix Metalloproteinase Activities"

    Article Title: Fragmentation of Fibronectin by Inherent Autolytic and Matrix Metalloproteinase Activities

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    doi: 10.1016/j.matbio.2010.09.004

    Interaction of FN with the carboxyl-terminal domain of MMP-2 Panel A. Interactions of FN with the carboxyl-terminal hemopexin-like domain of MMP-2 (PEX) were analyzed. FN was coated in 96 microwell plates (0.5 μg/well), blocked with BSA (10 mg/ml), and reacted with a concentration range (3 × 10 −6 – 10 −10 M) of biotinylated MMP-2, PEX, or MMP-2ΔPEX in which the PEX is deleted. Protein binding to coated FN was detected with alkaline phosphatase-conjugated streptavidin and PNPP substrate, and quantified at 405 nm. MMP-2 and PEX bound in a concentration-dependent saturable manner to FN. In comparison, MMP-2ΔPEX showed weak and non-saturable binding to this ligand. Panel B. The specific contribution of PEX to the binding was measured in competitive protein-protein binding assays in which biotinylated MMP-2 was added simultaneously with a concentration range of unlabeled MMP-2, PEX or ovalbumin. Like unlabeled MMP-2 (positive control), isolated PEX competitively inhibited the binding of MMP-2 to FN in a concentration-dependent manner pointing to PEX as an essential exosite for MMP-2 interactions with FN.
    Figure Legend Snippet: Interaction of FN with the carboxyl-terminal domain of MMP-2 Panel A. Interactions of FN with the carboxyl-terminal hemopexin-like domain of MMP-2 (PEX) were analyzed. FN was coated in 96 microwell plates (0.5 μg/well), blocked with BSA (10 mg/ml), and reacted with a concentration range (3 × 10 −6 – 10 −10 M) of biotinylated MMP-2, PEX, or MMP-2ΔPEX in which the PEX is deleted. Protein binding to coated FN was detected with alkaline phosphatase-conjugated streptavidin and PNPP substrate, and quantified at 405 nm. MMP-2 and PEX bound in a concentration-dependent saturable manner to FN. In comparison, MMP-2ΔPEX showed weak and non-saturable binding to this ligand. Panel B. The specific contribution of PEX to the binding was measured in competitive protein-protein binding assays in which biotinylated MMP-2 was added simultaneously with a concentration range of unlabeled MMP-2, PEX or ovalbumin. Like unlabeled MMP-2 (positive control), isolated PEX competitively inhibited the binding of MMP-2 to FN in a concentration-dependent manner pointing to PEX as an essential exosite for MMP-2 interactions with FN.

    Techniques Used: Concentration Assay, Protein Binding, Binding Assay, Positive Control, Isolation

    37) Product Images from "Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells"

    Article Title: Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00153.2008

    Surface biotinylation of NBCe1 and NBCn1 in ParC5 cells. A : surface biotinylation shows removal of the electrogenic NBCe1 from the cell surface. A-1 and A-3 : typical Western blots of the cell surface-biotinylated NBCe1 proteins. In A-1 , cells were untreated
    Figure Legend Snippet: Surface biotinylation of NBCe1 and NBCn1 in ParC5 cells. A : surface biotinylation shows removal of the electrogenic NBCe1 from the cell surface. A-1 and A-3 : typical Western blots of the cell surface-biotinylated NBCe1 proteins. In A-1 , cells were untreated

    Techniques Used: Western Blot

    38) Product Images from "TGFβ2-induced formation of lipid droplets supports acidosis-driven EMT and the metastatic spreading of cancer cells"

    Article Title: TGFβ2-induced formation of lipid droplets supports acidosis-driven EMT and the metastatic spreading of cancer cells

    Journal: Nature Communications

    doi: 10.1038/s41467-019-14262-3

    TGF-β2 promotes FA uptake and TG accumulation into LD. a – c Abundance of neutral lipids (NL), phospholipids (PL) and free fatty acids (FFA) ( a ), abundance of saturated and monounsaturated fatty acids (SFA and MUFA, respectively) in the neutral lipid fraction ( b ), and LD content ( c ) in native SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h. d , e 14 C-palmitate uptake for 10 min in SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h ( d ) and in acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days in absence or presence of 4 ng/ml TGF-β2 for 24 h ( e ). f – h Representative immunoblotting for cell surface-localized CD36 and total biotinylated proteins in native and acidosis-adapted SiHa cells following treatment with 4 ng/ml TGF-β2 for 6 h and 24 h ( f ), following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( g ) or with 4 ng/ml TGF-β2 and 10 µM PKC-ζ pseudo-substrate inhibitor for 24 h ( h ). i , j Quantification of surface-localized CD36 in native and acidosis-adapted SiHa cells treated as indicated in ( h ). k , l mRNA ( k ) and protein expression of DGAT1 ( l ) in native and acidosis-adapted tumor cells. m – o Representative immunoblotting for DGAT1 in native and acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( m ), with 10 µM PKC-ζ pseudo-substrate inhibitor for 24 h ( n ) or with 10 µM GW6471 for 48 h ( o ). p mRNA expression of PLIN2 in native and acidosis-adapted SiHa cells following treatment with 10 µM TGFβ2-specific antisense oligonucleotide Trabedersen for 7 days. q Co-expression analysis of TGFB2 , PLIN1 , PLIN2 , and PLIN3 genes in human healthy volunteers and colorectal cancer patient samples. Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by Student’s t -test ( c , d , j ), by one-way ANOVA ( e – i ) or two-way ANOVA ( a , k , p ) with Bonferroni multiple-comparison analysis. * p
    Figure Legend Snippet: TGF-β2 promotes FA uptake and TG accumulation into LD. a – c Abundance of neutral lipids (NL), phospholipids (PL) and free fatty acids (FFA) ( a ), abundance of saturated and monounsaturated fatty acids (SFA and MUFA, respectively) in the neutral lipid fraction ( b ), and LD content ( c ) in native SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h. d , e 14 C-palmitate uptake for 10 min in SiHa cells after treatment with 4 ng/ml TGF-β2 for 6 h ( d ) and in acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days in absence or presence of 4 ng/ml TGF-β2 for 24 h ( e ). f – h Representative immunoblotting for cell surface-localized CD36 and total biotinylated proteins in native and acidosis-adapted SiHa cells following treatment with 4 ng/ml TGF-β2 for 6 h and 24 h ( f ), following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( g ) or with 4 ng/ml TGF-β2 and 10 µM PKC-ζ pseudo-substrate inhibitor for 24 h ( h ). i , j Quantification of surface-localized CD36 in native and acidosis-adapted SiHa cells treated as indicated in ( h ). k , l mRNA ( k ) and protein expression of DGAT1 ( l ) in native and acidosis-adapted tumor cells. m – o Representative immunoblotting for DGAT1 in native and acidosis-adapted SiHa cells following treatment with 10 µM Trabedersen for 7 days or 2 µM SB431542 for 24 h ( m ), with 10 µM PKC-ζ pseudo-substrate inhibitor for 24 h ( n ) or with 10 µM GW6471 for 48 h ( o ). p mRNA expression of PLIN2 in native and acidosis-adapted SiHa cells following treatment with 10 µM TGFβ2-specific antisense oligonucleotide Trabedersen for 7 days. q Co-expression analysis of TGFB2 , PLIN1 , PLIN2 , and PLIN3 genes in human healthy volunteers and colorectal cancer patient samples. Data are represented as mean ± SEM of three independent experiments (with ≥6 technical replicates). Significance was determined by Student’s t -test ( c , d , j ), by one-way ANOVA ( e – i ) or two-way ANOVA ( a , k , p ) with Bonferroni multiple-comparison analysis. * p

    Techniques Used: Expressing

    39) Product Images from "Viral Protein Determinants of Lassa Virus Entry and Release from Polarized Epithelial Cells ▿"

    Article Title: Viral Protein Determinants of Lassa Virus Entry and Release from Polarized Epithelial Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02240-09

    Localization of Lassa virus glycoprotein GP in polarized epithelial cells. (A) MDCK-II cells were infected with LASV at an MOI of 1 from the basolateral cell surface. At 72 h p.i. cells were fixed with methanol-acetone and subsequently treated with 4% PFA for 48 h. The LASV glycoprotein subunit GP-1 (red) and the tight-junction resident protein ZO-3 (green) were immunostained. Distributions of GP and ZO-3 were monitored by using confocal laser scanning microscopy in the xy and yz directions. The xy scan is a composite of several images from the apical and middle regions of the image stack because of the irregular height of the cells. The scale bars denote 20 μm. (B) Immunodetection of GP in a stably GP-expressing nonpermeabilized MDCK-II cell line. Cells were fixed with 4% paraformaldehyde and immunostained using an α-GP-2-N antibody (red). The scale bars denote 20 μm. (C) Proteins on the cell surface of stably LASV GP- and VSV G-expressing MDCK-II cells were labeled with sulfo-NHS-biotin on either the apical or basolateral side. After cell lysis, biotinylated proteins were precipitated using streptavidin-coupled Sepharose beads and subsequently subjected to SDS-PAGE and immunoblot analysis using α-GP-2-C or α-VSV G antiserum.
    Figure Legend Snippet: Localization of Lassa virus glycoprotein GP in polarized epithelial cells. (A) MDCK-II cells were infected with LASV at an MOI of 1 from the basolateral cell surface. At 72 h p.i. cells were fixed with methanol-acetone and subsequently treated with 4% PFA for 48 h. The LASV glycoprotein subunit GP-1 (red) and the tight-junction resident protein ZO-3 (green) were immunostained. Distributions of GP and ZO-3 were monitored by using confocal laser scanning microscopy in the xy and yz directions. The xy scan is a composite of several images from the apical and middle regions of the image stack because of the irregular height of the cells. The scale bars denote 20 μm. (B) Immunodetection of GP in a stably GP-expressing nonpermeabilized MDCK-II cell line. Cells were fixed with 4% paraformaldehyde and immunostained using an α-GP-2-N antibody (red). The scale bars denote 20 μm. (C) Proteins on the cell surface of stably LASV GP- and VSV G-expressing MDCK-II cells were labeled with sulfo-NHS-biotin on either the apical or basolateral side. After cell lysis, biotinylated proteins were precipitated using streptavidin-coupled Sepharose beads and subsequently subjected to SDS-PAGE and immunoblot analysis using α-GP-2-C or α-VSV G antiserum.

    Techniques Used: Infection, Confocal Laser Scanning Microscopy, Immunodetection, Stable Transfection, Expressing, Labeling, Lysis, SDS Page

    40) Product Images from "A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR)"

    Article Title: A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR)

    Journal: Journal of Virology

    doi: 10.1128/JVI.00763-17

    Biochemical validation of interactions between arenavirus NPs expressed from plasmids and endogenous host proteins. (A) HEK 293T cells were cotransfected with a plasmid encoding the respective arenavirus NP with a C-terminal HA epitope tag, the TEV cleavage site, and a biotin acceptor peptide, along with a second plasmid that encodes BirA, a bacterial biotin ligase, to ensure biotinylation of the viral NPs. As a control, cells were cotransfected with the BirA plasmid and an empty vector (p0). Biotinylated NPs and associated host proteins were affinity purified from cell lysates (input) by use of magnetic streptavidin beads, and captured proteins were detected by Western blotting. (B and C) HEK 293T cells were transfected with a plasmid encoding the respective arenavirus NP with a C-terminal HA epitope tag, the TEV cleavage site, and a biotin acceptor peptide or, as a control, an empty vector (p0). (B) PKR (bait) was immunoprecipitated with a monoclonal antibody to the C terminus of PKR or with an irrelevant rabbit IgG. PKR (bait) and the associated viral NP (prey) were detected by Western blotting. (C) AIFM1 (bait) was immunoprecipitated with a polyclonal antibody. AIFM1 (bait) and the associated viral NP (prey) were detected by Western blotting. Data are representative of 2 (A), 3 (B), or 2 (C) independent experiments.
    Figure Legend Snippet: Biochemical validation of interactions between arenavirus NPs expressed from plasmids and endogenous host proteins. (A) HEK 293T cells were cotransfected with a plasmid encoding the respective arenavirus NP with a C-terminal HA epitope tag, the TEV cleavage site, and a biotin acceptor peptide, along with a second plasmid that encodes BirA, a bacterial biotin ligase, to ensure biotinylation of the viral NPs. As a control, cells were cotransfected with the BirA plasmid and an empty vector (p0). Biotinylated NPs and associated host proteins were affinity purified from cell lysates (input) by use of magnetic streptavidin beads, and captured proteins were detected by Western blotting. (B and C) HEK 293T cells were transfected with a plasmid encoding the respective arenavirus NP with a C-terminal HA epitope tag, the TEV cleavage site, and a biotin acceptor peptide or, as a control, an empty vector (p0). (B) PKR (bait) was immunoprecipitated with a monoclonal antibody to the C terminus of PKR or with an irrelevant rabbit IgG. PKR (bait) and the associated viral NP (prey) were detected by Western blotting. (C) AIFM1 (bait) was immunoprecipitated with a polyclonal antibody. AIFM1 (bait) and the associated viral NP (prey) were detected by Western blotting. Data are representative of 2 (A), 3 (B), or 2 (C) independent experiments.

    Techniques Used: Plasmid Preparation, Affinity Purification, Western Blot, Transfection, Immunoprecipitation

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    Article Snippet: .. Extracts were clarified by centrifugation (20,000×g for 15 min), and biotinylated proteins were isolated by immobilization on streptavidin-conjugated Sepharose beads (Pierce). .. Washed beads were eluted with LDS sample buffer before resolving by SDS-PAGE, transferred to nitrocellulose membranes, and probed for FLAG-tagged receptor (M1 antibody; Sigma).

    Article Title: Asp433 in the closing gate of ASIC1 determines stability of the open state without changing properties of the selectivity filter or Ca2+ block
    Article Snippet: .. After lysis of cells with 1% Triton X-100 and centrifugation at 8,000 g for removal of the yolk, biotinylated proteins were recovered with streptavidin beads (Thermo Fisher Scientific). ..

    Isolation:

    Article Title: Dysbindin Promotes the Post-Endocytic Sorting of G Protein-Coupled Receptors to Lysosomes
    Article Snippet: .. Extracts were clarified by centrifugation (20,000×g for 15 min), and biotinylated proteins were isolated by immobilization on streptavidin-conjugated Sepharose beads (Pierce). .. Washed beads were eluted with LDS sample buffer before resolving by SDS-PAGE, transferred to nitrocellulose membranes, and probed for FLAG-tagged receptor (M1 antibody; Sigma).

    Purification:

    Article Title: Hsp70 Chaperones and Type I PRMTs Are Sequestered at Intranuclear Inclusions Caused by Polyalanine Expansions in PABPN1
    Article Snippet: .. Pull-down assays with biotinylated proteins The normal and expanded forms of recombinant PABPN1 purified from insect cells (see for details) were biotinylated using EZ-Link PEO-Maleimide Activated Biotin (Pierce), according to the manufacturer's instructions. .. Buffer exchange to remove unincorporated PEO-biotin was performed using NAP-5 or NAP-10 columns (Pharmacia Biotech).

    Concentration Assay:

    Article Title: Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein
    Article Snippet: .. The concentration of biotinylated proteins was determined using the NanoDrop 2000 system (ThermoScientific). .. The biotinylation efficiency of proteins was qualitatively evaluated using dot-blot analysis.

    Incubation:

    Article Title: Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response
    Article Snippet: .. Hence, uninfected RAW cells were incubated with either the biotinylated WP1130 (WP) or the inactive biotinylated analog (Null), lysed, and biotinylated proteins precipitated with streptavidin agarose beads (Invitrogen). .. Proteins were resolved by SDS-Page and stained with Sypro Ruby Red (Invitrogen) ( ).

    Western Blot:

    Article Title: Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions
    Article Snippet: .. Biotinylated proteins were pulled down with NeutrAvidin-linked beads (Pierce) and detected by Western blotting. ..

    Lysis:

    Article Title: Asp433 in the closing gate of ASIC1 determines stability of the open state without changing properties of the selectivity filter or Ca2+ block
    Article Snippet: .. After lysis of cells with 1% Triton X-100 and centrifugation at 8,000 g for removal of the yolk, biotinylated proteins were recovered with streptavidin beads (Thermo Fisher Scientific). ..

    Recombinant:

    Article Title: Hsp70 Chaperones and Type I PRMTs Are Sequestered at Intranuclear Inclusions Caused by Polyalanine Expansions in PABPN1
    Article Snippet: .. Pull-down assays with biotinylated proteins The normal and expanded forms of recombinant PABPN1 purified from insect cells (see for details) were biotinylated using EZ-Link PEO-Maleimide Activated Biotin (Pierce), according to the manufacturer's instructions. .. Buffer exchange to remove unincorporated PEO-biotin was performed using NAP-5 or NAP-10 columns (Pharmacia Biotech).

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  • 93
    Thermo Fisher biotinylated rna pull down assays t98g
    Determination of the EV71 IRES sequences required for the binding of PTB. A Prediction of the <t>RNA</t> secondary structure of the EV71 5′UTR by the mfold web serve. The modified schematic representation of the secondary structure of poliovirus (PV) 5′UTR (modified from Hellen et al . 1994 ) is shown in the top right corner. B Plasmids carrying different deletions in the five stem-loops of EV71 IRES: pGEM-3zf-(SL II, 121–181), (SL III, 190–230), (SL IV, 241–450), (SL V, 451–463), (SL VI, 564–742). C Analysis of the regions responsible for the interaction in the EV71 IRES region using various truncated RNA forms, transcribed in vitro and <t>biotinylated.</t> <t>T98G</t> cell lysate were incubated with these biotin-labeled RNAs and the non-biotinylated IRES RNA probes were used as controls. After being pulled down by streptavidin, the protein complex was separated by SDS-PAGE and Western blot was carried out to detect PTB in the pulled-down complex (lanes 1–7).
    Biotinylated Rna Pull Down Assays T98g, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated rna pull down assays t98g/product/Thermo Fisher
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    89
    Thermo Fisher biotinylated cell surface proteins cell surface proteins
    Dopamine reduced turnover and solubility of PrP C . Cell surface proteins was <t>biotinylated</t> and chased at time zero (input) or after 1 h incubation in the presence or absence of 50 μM of dopamine. After 1 h, dopamine treated cells showed higher amount of remaining PrP C compared to untreated cells (A) . After dopamine treatment, insoluble proteins in sarkosyl 1% were recovered by ultracentrifugation. Higher amounts of PrP C were recovered in pellet fractions and lower amount in supernatants (SN), indicating lower solubility (B) . Mean band intensity of three independent experiments was plotted with respective CI95. Dark gray bars represent SN and light gray bars represent pellet quantification (B) . * no overlap of CI95 between indicated group and control group (0 μM). rPrP (10 μg) was incubated with designated concentration of dopamine (DA) for 24 h or with 400 μM of dopamine for indicated times (C) . When 400 μM of metabisulfite (#) was added, the oligomerization was prevented (C) . rPrP monomer migrated close to 26 kDa marker. Three independent experiments were performed and representative images are shown.
    Biotinylated Cell Surface Proteins Cell Surface Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated cell surface proteins cell surface proteins/product/Thermo Fisher
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    biotinylated cell surface proteins cell surface proteins - by Bioz Stars, 2020-07
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    88
    Thermo Fisher non biotinylated immunoprecipitated protein
    Sera from BNP dams recognize MHC class I and non-MHC class I targets. Cell surface proteins of wild-type MDBK (WT), B2M KO MDBK (B2M KO) or PBLk were <t>biotinylated</t> and subsequently stained with sera of Pregsure© BVD-vaccinated BNP dams (BNP+Vacc+) or control dams not vaccinated with Pregsure© BVD (BNP−Vacc−). After washing away unbound Abs, cells were lysed. Abs and bound Ag were precipitated using Protein G coupled dynabeads, separated by non-reducing gel electrophoresis and blotted on nitrocellulose membrane. ( a ) WT and KO MDBK cells were stained with serum from a BNP+Vacc+ dam or a BNP−Vacc− dam. Cell surface proteins were visualized using SA-AP. ( b ) As in A, but <t>immunoprecipitated</t> MHC class I was visualized using anti-bovine MHC class I mAb ILA88. ( c ) B2M KO MDBK cells were stained with sera from BNP+Vacc+ dams (n = 7) and BNP−Vacc− dams (n = 2). Cell surface proteins were visualized using SA-AP. The sera from BNP+Vacc+ dams were ranked according to alloantibody staining intensity of B2M KO MDBK cells as in Fig. 3c . Representative for two separate experiments. ( d ) PBLk from a healthy donor were stained with sera from BNP+Vacc+ dams (n = 2) or a BNP−Vacc− dam. Cell surface proteins and MHC I were visualized using SA-AP and mAb ILA88, respectively. Representative for two separate experiments with different PBLk donors. MW = Molecular weight markers (kDa). ITGB1 = integrin β1. ITGA3 = integrin α3. * = nonspecific signal. Boxes in C indicate regions that were subjected to mass spectrometry analysis.
    Non Biotinylated Immunoprecipitated Protein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher biotinylated proteins
    Relative magnitude of whole cell currents evoked by pH 6.0 of mutants in positions Gly-432 and Asp-433. (A) Normalized currents of Gly-432 substitutions. Columns are the average of ≥12 cells from three different preparations. Error bars are standard deviations. Asterisks indicate significant difference from G432 by unpaired t test, P ≤ 0.005. (B) Western blot of lASIC1 detected with anti-FLAG monoclonal antibody from eight oocytes injected with 5 ng cRNA that were <t>biotinylated</t> after measurements of whole cell currents by TEVC. (C) Normalized currents of substitutions of Asp-433. (D) Western blot of oocytes as shown in C, but oocytes expressing N433 and G433 were injected with 50 ng cRNA.
    Biotinylated Proteins, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Determination of the EV71 IRES sequences required for the binding of PTB. A Prediction of the RNA secondary structure of the EV71 5′UTR by the mfold web serve. The modified schematic representation of the secondary structure of poliovirus (PV) 5′UTR (modified from Hellen et al . 1994 ) is shown in the top right corner. B Plasmids carrying different deletions in the five stem-loops of EV71 IRES: pGEM-3zf-(SL II, 121–181), (SL III, 190–230), (SL IV, 241–450), (SL V, 451–463), (SL VI, 564–742). C Analysis of the regions responsible for the interaction in the EV71 IRES region using various truncated RNA forms, transcribed in vitro and biotinylated. T98G cell lysate were incubated with these biotin-labeled RNAs and the non-biotinylated IRES RNA probes were used as controls. After being pulled down by streptavidin, the protein complex was separated by SDS-PAGE and Western blot was carried out to detect PTB in the pulled-down complex (lanes 1–7).

    Journal: Virologica Sinica

    Article Title: Polypyrimidine Tract-Binding Protein Regulates Enterovirus 71 Translation Through Interaction with the Internal Ribosomal Entry Site

    doi: 10.1007/s12250-019-00089-1

    Figure Lengend Snippet: Determination of the EV71 IRES sequences required for the binding of PTB. A Prediction of the RNA secondary structure of the EV71 5′UTR by the mfold web serve. The modified schematic representation of the secondary structure of poliovirus (PV) 5′UTR (modified from Hellen et al . 1994 ) is shown in the top right corner. B Plasmids carrying different deletions in the five stem-loops of EV71 IRES: pGEM-3zf-(SL II, 121–181), (SL III, 190–230), (SL IV, 241–450), (SL V, 451–463), (SL VI, 564–742). C Analysis of the regions responsible for the interaction in the EV71 IRES region using various truncated RNA forms, transcribed in vitro and biotinylated. T98G cell lysate were incubated with these biotin-labeled RNAs and the non-biotinylated IRES RNA probes were used as controls. After being pulled down by streptavidin, the protein complex was separated by SDS-PAGE and Western blot was carried out to detect PTB in the pulled-down complex (lanes 1–7).

    Article Snippet: Biotinylated RNA Pull-Down Assays T98G, HA, RD, and SH-SY5Y cells were pelleted, and then prepared using nuclear protein extraction reagents (Thermo, USA).

    Techniques: Binding Assay, Modification, In Vitro, Incubation, Labeling, SDS Page, Western Blot

    Interaction of PTB with the EV71 IRES region. A Pull down of proteins with the EV71 IRES in T98G cell lysate. The specific bands interacting with EV71 IRES are shown by silver staining. Lane 1: biotinylated IRES RNA, Lane 2: nonbiotinylated EV71 IRES RNA. B A specific association between PTB and the EV71 IRES region was confirmed by Western blotting and a competition assay. Increasing amounts of unlabelled RNA were added to compete with the biotin-labeled EV71 IRES RNA interacting with PTB. The eluted proteins were separated by 12% SDS-PAGE. Lanes are as follows: lanes 1–4, unlabeled EV71 IRES RNA; lanes 5–8, unlabeled yeast tRNA. C Extracts of RD cells, SH-SY5Y cells, and HA cells were prepared and then incubated without RNA (lane 2), with biotinylated actin RNA (lane 3), non-biotinylated EV71 IRES RNA (lane 4), or biotinylated EV71 IRES RNA (lane 5). After pull-down assay, the bound proteins were eluted, boiled, and subjected to 12% SDS-PAGE. PTB protein was detected by western blot with a rabbit anti-PTB antibody. The inputs were cell extracts of RD, SH-SY5Y and HA (lane 1). D EV71 IRES RNA was pulled down with PTB from EV71-infected T98G cell lysate. T98G cells were infected with EV71 at an MOI of 20 for 6 h and then cell extracts were incubated with rabbit anti-PTB antibody (lanes 2 and 6), normal rabbit IgG (lanes 3 and 7), or without antibody (− Ab) (lanes 4 and 8). Following washing and dissociation, the RNA extract was prepared and subjected to RT-PCR analysis with primers specific for the ribosomal protein S16 (PRS16) RNA (lanes1–4) or for EV71 IRES region RNA (lanes 5–8). Lane 1, cell lysate without immunoprecipitation as a positive RT-PCR control; Lane 2, anti-PTB antibody incubated with 200 mg infected-cell lysate; Lane 3, negative control with rabbit IgG; Lane 4, negative control with no antibody.

    Journal: Virologica Sinica

    Article Title: Polypyrimidine Tract-Binding Protein Regulates Enterovirus 71 Translation Through Interaction with the Internal Ribosomal Entry Site

    doi: 10.1007/s12250-019-00089-1

    Figure Lengend Snippet: Interaction of PTB with the EV71 IRES region. A Pull down of proteins with the EV71 IRES in T98G cell lysate. The specific bands interacting with EV71 IRES are shown by silver staining. Lane 1: biotinylated IRES RNA, Lane 2: nonbiotinylated EV71 IRES RNA. B A specific association between PTB and the EV71 IRES region was confirmed by Western blotting and a competition assay. Increasing amounts of unlabelled RNA were added to compete with the biotin-labeled EV71 IRES RNA interacting with PTB. The eluted proteins were separated by 12% SDS-PAGE. Lanes are as follows: lanes 1–4, unlabeled EV71 IRES RNA; lanes 5–8, unlabeled yeast tRNA. C Extracts of RD cells, SH-SY5Y cells, and HA cells were prepared and then incubated without RNA (lane 2), with biotinylated actin RNA (lane 3), non-biotinylated EV71 IRES RNA (lane 4), or biotinylated EV71 IRES RNA (lane 5). After pull-down assay, the bound proteins were eluted, boiled, and subjected to 12% SDS-PAGE. PTB protein was detected by western blot with a rabbit anti-PTB antibody. The inputs were cell extracts of RD, SH-SY5Y and HA (lane 1). D EV71 IRES RNA was pulled down with PTB from EV71-infected T98G cell lysate. T98G cells were infected with EV71 at an MOI of 20 for 6 h and then cell extracts were incubated with rabbit anti-PTB antibody (lanes 2 and 6), normal rabbit IgG (lanes 3 and 7), or without antibody (− Ab) (lanes 4 and 8). Following washing and dissociation, the RNA extract was prepared and subjected to RT-PCR analysis with primers specific for the ribosomal protein S16 (PRS16) RNA (lanes1–4) or for EV71 IRES region RNA (lanes 5–8). Lane 1, cell lysate without immunoprecipitation as a positive RT-PCR control; Lane 2, anti-PTB antibody incubated with 200 mg infected-cell lysate; Lane 3, negative control with rabbit IgG; Lane 4, negative control with no antibody.

    Article Snippet: Biotinylated RNA Pull-Down Assays T98G, HA, RD, and SH-SY5Y cells were pelleted, and then prepared using nuclear protein extraction reagents (Thermo, USA).

    Techniques: Silver Staining, Western Blot, Competitive Binding Assay, Labeling, SDS Page, Incubation, Pull Down Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Negative Control

    Dopamine reduced turnover and solubility of PrP C . Cell surface proteins was biotinylated and chased at time zero (input) or after 1 h incubation in the presence or absence of 50 μM of dopamine. After 1 h, dopamine treated cells showed higher amount of remaining PrP C compared to untreated cells (A) . After dopamine treatment, insoluble proteins in sarkosyl 1% were recovered by ultracentrifugation. Higher amounts of PrP C were recovered in pellet fractions and lower amount in supernatants (SN), indicating lower solubility (B) . Mean band intensity of three independent experiments was plotted with respective CI95. Dark gray bars represent SN and light gray bars represent pellet quantification (B) . * no overlap of CI95 between indicated group and control group (0 μM). rPrP (10 μg) was incubated with designated concentration of dopamine (DA) for 24 h or with 400 μM of dopamine for indicated times (C) . When 400 μM of metabisulfite (#) was added, the oligomerization was prevented (C) . rPrP monomer migrated close to 26 kDa marker. Three independent experiments were performed and representative images are shown.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Dopamine induces the accumulation of insoluble prion protein and affects autophagic flux

    doi: 10.3389/fncel.2015.00012

    Figure Lengend Snippet: Dopamine reduced turnover and solubility of PrP C . Cell surface proteins was biotinylated and chased at time zero (input) or after 1 h incubation in the presence or absence of 50 μM of dopamine. After 1 h, dopamine treated cells showed higher amount of remaining PrP C compared to untreated cells (A) . After dopamine treatment, insoluble proteins in sarkosyl 1% were recovered by ultracentrifugation. Higher amounts of PrP C were recovered in pellet fractions and lower amount in supernatants (SN), indicating lower solubility (B) . Mean band intensity of three independent experiments was plotted with respective CI95. Dark gray bars represent SN and light gray bars represent pellet quantification (B) . * no overlap of CI95 between indicated group and control group (0 μM). rPrP (10 μg) was incubated with designated concentration of dopamine (DA) for 24 h or with 400 μM of dopamine for indicated times (C) . When 400 μM of metabisulfite (#) was added, the oligomerization was prevented (C) . rPrP monomer migrated close to 26 kDa marker. Three independent experiments were performed and representative images are shown.

    Article Snippet: Purification of biotinylated cell surface proteins Cell surface proteins were biotinylated using Pierce Cell Surface Protein Isolation Kit (Thermo Scientific).

    Techniques: Solubility, Incubation, Concentration Assay, Marker

    Sera from BNP dams recognize MHC class I and non-MHC class I targets. Cell surface proteins of wild-type MDBK (WT), B2M KO MDBK (B2M KO) or PBLk were biotinylated and subsequently stained with sera of Pregsure© BVD-vaccinated BNP dams (BNP+Vacc+) or control dams not vaccinated with Pregsure© BVD (BNP−Vacc−). After washing away unbound Abs, cells were lysed. Abs and bound Ag were precipitated using Protein G coupled dynabeads, separated by non-reducing gel electrophoresis and blotted on nitrocellulose membrane. ( a ) WT and KO MDBK cells were stained with serum from a BNP+Vacc+ dam or a BNP−Vacc− dam. Cell surface proteins were visualized using SA-AP. ( b ) As in A, but immunoprecipitated MHC class I was visualized using anti-bovine MHC class I mAb ILA88. ( c ) B2M KO MDBK cells were stained with sera from BNP+Vacc+ dams (n = 7) and BNP−Vacc− dams (n = 2). Cell surface proteins were visualized using SA-AP. The sera from BNP+Vacc+ dams were ranked according to alloantibody staining intensity of B2M KO MDBK cells as in Fig. 3c . Representative for two separate experiments. ( d ) PBLk from a healthy donor were stained with sera from BNP+Vacc+ dams (n = 2) or a BNP−Vacc− dam. Cell surface proteins and MHC I were visualized using SA-AP and mAb ILA88, respectively. Representative for two separate experiments with different PBLk donors. MW = Molecular weight markers (kDa). ITGB1 = integrin β1. ITGA3 = integrin α3. * = nonspecific signal. Boxes in C indicate regions that were subjected to mass spectrometry analysis.

    Journal: Scientific Reports

    Article Title: Pathogenicity of Bovine Neonatal Pancytopenia-associated vaccine-induced alloantibodies correlates with Major Histocompatibility Complex class I expression

    doi: 10.1038/srep12748

    Figure Lengend Snippet: Sera from BNP dams recognize MHC class I and non-MHC class I targets. Cell surface proteins of wild-type MDBK (WT), B2M KO MDBK (B2M KO) or PBLk were biotinylated and subsequently stained with sera of Pregsure© BVD-vaccinated BNP dams (BNP+Vacc+) or control dams not vaccinated with Pregsure© BVD (BNP−Vacc−). After washing away unbound Abs, cells were lysed. Abs and bound Ag were precipitated using Protein G coupled dynabeads, separated by non-reducing gel electrophoresis and blotted on nitrocellulose membrane. ( a ) WT and KO MDBK cells were stained with serum from a BNP+Vacc+ dam or a BNP−Vacc− dam. Cell surface proteins were visualized using SA-AP. ( b ) As in A, but immunoprecipitated MHC class I was visualized using anti-bovine MHC class I mAb ILA88. ( c ) B2M KO MDBK cells were stained with sera from BNP+Vacc+ dams (n = 7) and BNP−Vacc− dams (n = 2). Cell surface proteins were visualized using SA-AP. The sera from BNP+Vacc+ dams were ranked according to alloantibody staining intensity of B2M KO MDBK cells as in Fig. 3c . Representative for two separate experiments. ( d ) PBLk from a healthy donor were stained with sera from BNP+Vacc+ dams (n = 2) or a BNP−Vacc− dam. Cell surface proteins and MHC I were visualized using SA-AP and mAb ILA88, respectively. Representative for two separate experiments with different PBLk donors. MW = Molecular weight markers (kDa). ITGB1 = integrin β1. ITGA3 = integrin α3. * = nonspecific signal. Boxes in C indicate regions that were subjected to mass spectrometry analysis.

    Article Snippet: Non-biotinylated immunoprecipitated protein samples were processed in parallel and visualized using GelCode Blue Stain Reagent (Thermo Scientific) after PAGE.

    Techniques: Staining, Nucleic Acid Electrophoresis, Immunoprecipitation, Molecular Weight, Mass Spectrometry

    Relative magnitude of whole cell currents evoked by pH 6.0 of mutants in positions Gly-432 and Asp-433. (A) Normalized currents of Gly-432 substitutions. Columns are the average of ≥12 cells from three different preparations. Error bars are standard deviations. Asterisks indicate significant difference from G432 by unpaired t test, P ≤ 0.005. (B) Western blot of lASIC1 detected with anti-FLAG monoclonal antibody from eight oocytes injected with 5 ng cRNA that were biotinylated after measurements of whole cell currents by TEVC. (C) Normalized currents of substitutions of Asp-433. (D) Western blot of oocytes as shown in C, but oocytes expressing N433 and G433 were injected with 50 ng cRNA.

    Journal: The Journal of General Physiology

    Article Title: Asp433 in the closing gate of ASIC1 determines stability of the open state without changing properties of the selectivity filter or Ca2+ block

    doi: 10.1085/jgp.201010576

    Figure Lengend Snippet: Relative magnitude of whole cell currents evoked by pH 6.0 of mutants in positions Gly-432 and Asp-433. (A) Normalized currents of Gly-432 substitutions. Columns are the average of ≥12 cells from three different preparations. Error bars are standard deviations. Asterisks indicate significant difference from G432 by unpaired t test, P ≤ 0.005. (B) Western blot of lASIC1 detected with anti-FLAG monoclonal antibody from eight oocytes injected with 5 ng cRNA that were biotinylated after measurements of whole cell currents by TEVC. (C) Normalized currents of substitutions of Asp-433. (D) Western blot of oocytes as shown in C, but oocytes expressing N433 and G433 were injected with 50 ng cRNA.

    Article Snippet: After lysis of cells with 1% Triton X-100 and centrifugation at 8,000 g for removal of the yolk, biotinylated proteins were recovered with streptavidin beads (Thermo Fisher Scientific).

    Techniques: Western Blot, Injection, Expressing