anti flag mab  (Millipore)


Bioz Verified Symbol Millipore is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99

    Structured Review

    Millipore anti flag mab
    Most alanine H mutants spanning the “spacer” <t>CDV</t> H-stalk region are fusion-promotion impaired. (A) Schematic representation of the main functional domains of the full length CDV H-wt protein. The detailed primary sequence of the “spacer” section and the residues defining mAb αH-1347 epitope are shown. The H stalk-section scanned by alanine mutagenesis is also highlighted. (B) Syncytium formation assay. Cell-to-cell fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H-wt or derived alanine variants with CDV F-wt. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (C) Characterization of H mutants. Standard and derivative H mutants were expressed in Vero cells. One day post-transfection, cell surface expression (recorded by <t>anti-FLAG</t> staining followed by flow cytometry analyses), SLAM binding efficiency (calculated as described in the legend of Fig 1D , but without addition of mAbs before SLAM treatments) and fusion activity (monitored as described in the legend of Fig 1B ) were determined. All values were normalized to H-wt. (D) Reactivity of standard and derivative H mutants to mAb αH-1347. MFI values were recorded by flow cytometry 24h post-transfection in Vero cells. (E) Effect of SLAM treatment on mAb αH-1347’s H-binding activity (37°C). The assay and values were determined as described in the legend of Fig 4D . Means ± S.D. of data from four independent experiments performed in triplicates are shown. To determine the statistical significance of differences between the standard and mutant H (127 and 138) data sets, unpaired two-tailed t tests were performed (*, P
    Anti Flag Mab, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag mab/product/Millipore
    Average 99 stars, based on 16 article reviews
    Price from $9.99 to $1999.99
    anti flag mab - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Sequential Conformational Changes in the Morbillivirus Attachment Protein Initiate the Membrane Fusion Process"

    Article Title: Sequential Conformational Changes in the Morbillivirus Attachment Protein Initiate the Membrane Fusion Process

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004880

    Most alanine H mutants spanning the “spacer” CDV H-stalk region are fusion-promotion impaired. (A) Schematic representation of the main functional domains of the full length CDV H-wt protein. The detailed primary sequence of the “spacer” section and the residues defining mAb αH-1347 epitope are shown. The H stalk-section scanned by alanine mutagenesis is also highlighted. (B) Syncytium formation assay. Cell-to-cell fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H-wt or derived alanine variants with CDV F-wt. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (C) Characterization of H mutants. Standard and derivative H mutants were expressed in Vero cells. One day post-transfection, cell surface expression (recorded by anti-FLAG staining followed by flow cytometry analyses), SLAM binding efficiency (calculated as described in the legend of Fig 1D , but without addition of mAbs before SLAM treatments) and fusion activity (monitored as described in the legend of Fig 1B ) were determined. All values were normalized to H-wt. (D) Reactivity of standard and derivative H mutants to mAb αH-1347. MFI values were recorded by flow cytometry 24h post-transfection in Vero cells. (E) Effect of SLAM treatment on mAb αH-1347’s H-binding activity (37°C). The assay and values were determined as described in the legend of Fig 4D . Means ± S.D. of data from four independent experiments performed in triplicates are shown. To determine the statistical significance of differences between the standard and mutant H (127 and 138) data sets, unpaired two-tailed t tests were performed (*, P
    Figure Legend Snippet: Most alanine H mutants spanning the “spacer” CDV H-stalk region are fusion-promotion impaired. (A) Schematic representation of the main functional domains of the full length CDV H-wt protein. The detailed primary sequence of the “spacer” section and the residues defining mAb αH-1347 epitope are shown. The H stalk-section scanned by alanine mutagenesis is also highlighted. (B) Syncytium formation assay. Cell-to-cell fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H-wt or derived alanine variants with CDV F-wt. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (C) Characterization of H mutants. Standard and derivative H mutants were expressed in Vero cells. One day post-transfection, cell surface expression (recorded by anti-FLAG staining followed by flow cytometry analyses), SLAM binding efficiency (calculated as described in the legend of Fig 1D , but without addition of mAbs before SLAM treatments) and fusion activity (monitored as described in the legend of Fig 1B ) were determined. All values were normalized to H-wt. (D) Reactivity of standard and derivative H mutants to mAb αH-1347. MFI values were recorded by flow cytometry 24h post-transfection in Vero cells. (E) Effect of SLAM treatment on mAb αH-1347’s H-binding activity (37°C). The assay and values were determined as described in the legend of Fig 4D . Means ± S.D. of data from four independent experiments performed in triplicates are shown. To determine the statistical significance of differences between the standard and mutant H (127 and 138) data sets, unpaired two-tailed t tests were performed (*, P

    Techniques Used: Functional Assay, Sequencing, Mutagenesis, Tube Formation Assay, Activity Assay, Expressing, Derivative Assay, Transfection, Staining, Flow Cytometry, Cytometry, Binding Assay, Two Tailed Test

    Inhibition of headless H/F-mediated membrane fusion by mAb αH-1347. (A, C and D) Syncytium formation assay. Cell-to-cell fusion activity in Vero or Vero-cSLAM cells triggered by co-expression of CDV H-wt or headless H variants with CDV F-wt or CDV F-V447T (A75/17 strain) [ 62 ] in the presence (+) of absence of mAb αH-1347 (-). Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (B) F-triggering assay. The CDV F-V447T mutant was expressed in Vero cells together with the indicated Hstalk variants. One day post-transfection, the conformation of the FLAG-tagged F-V447T mutant was monitored by probing its reactivity with an anti-F prefusion-specific mAb (4941; green histograms), triggered-specific mAb (3633; red histograms) [ 61 , 63 ]. Secondary antibodies were added at 4°C, and to record quantitative values, mean fluorescence intensities were monitored by flow cytometry. (E) Quantitative fusion assays were performed as described in the legend of Fig 1B in the presence (light grey histograms) or absence (dark grey histograms) of mAb αH-1347. Means ± S.D. of data from three independent experiments performed in duplicates are shown.
    Figure Legend Snippet: Inhibition of headless H/F-mediated membrane fusion by mAb αH-1347. (A, C and D) Syncytium formation assay. Cell-to-cell fusion activity in Vero or Vero-cSLAM cells triggered by co-expression of CDV H-wt or headless H variants with CDV F-wt or CDV F-V447T (A75/17 strain) [ 62 ] in the presence (+) of absence of mAb αH-1347 (-). Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (B) F-triggering assay. The CDV F-V447T mutant was expressed in Vero cells together with the indicated Hstalk variants. One day post-transfection, the conformation of the FLAG-tagged F-V447T mutant was monitored by probing its reactivity with an anti-F prefusion-specific mAb (4941; green histograms), triggered-specific mAb (3633; red histograms) [ 61 , 63 ]. Secondary antibodies were added at 4°C, and to record quantitative values, mean fluorescence intensities were monitored by flow cytometry. (E) Quantitative fusion assays were performed as described in the legend of Fig 1B in the presence (light grey histograms) or absence (dark grey histograms) of mAb αH-1347. Means ± S.D. of data from three independent experiments performed in duplicates are shown.

    Techniques Used: Inhibition, Tube Formation Assay, Activity Assay, Expressing, Transfection, Mutagenesis, Fluorescence, Flow Cytometry, Cytometry

    mAb αH-1347 epitope mapping. (A) Schematic representation of the full length CDV-H-protein (H-wt) and a derivative soluble form (sH-ecto). The main functional regions of the stalk are color-coded. CT: cytosolic tail; TM: transmembrane domain. (B) Schematic representation of soluble Hstalk-RFP chimeric proteins and derivative deleted mutants (1–5). Del: deletion; RFP: red fluorescent protein. Drawings are not to scale. (C, D and F) Immunoprecipitation experiments performed with the indicated mAb of soluble H forms derived from the supernatant of transfected 293T cells. Antigenic materials were detected using polyclonal anti-GCN4 (upper panel) or anti-His antibody (lower panel). IP: immunoprecipitation; IB: immunoblotting. (E) Upper part: Schematic representation of the deletion 5 soluble Hstalk-RFP. Lower part: alignment of the primary amino acid sequence of the CDV H-stalk “spacer” section and the derivative triple alanine-scan mutants. Alanines are highlighted in red. (G) Homology structural model of CDV-F and positions (in red) where the mAb αH-1347 epitope has been inserted. Lower part: primary amino acid sequence of the CDV H-stalk “spacer” section with the encompassed F-transferred mAb-1347 peptide (highlighted in red). (H) Reactivities of F-wt-tagFLAG or F-wt-tag1347 with the conformation-insensitive anti-FLAG, the F prefusion state-specific anti-F-Pre (4941) [ 61 , 63 ] or the anti-CDV-H 1347 mAbs, probed 24 hours post-transfection in receptor-negative Vero cells. After addition of the secondary antibody, MFI values were recorded by flow cytometry analyses. Means ± S.D. of data from three independent experiments in triplicate are shown.
    Figure Legend Snippet: mAb αH-1347 epitope mapping. (A) Schematic representation of the full length CDV-H-protein (H-wt) and a derivative soluble form (sH-ecto). The main functional regions of the stalk are color-coded. CT: cytosolic tail; TM: transmembrane domain. (B) Schematic representation of soluble Hstalk-RFP chimeric proteins and derivative deleted mutants (1–5). Del: deletion; RFP: red fluorescent protein. Drawings are not to scale. (C, D and F) Immunoprecipitation experiments performed with the indicated mAb of soluble H forms derived from the supernatant of transfected 293T cells. Antigenic materials were detected using polyclonal anti-GCN4 (upper panel) or anti-His antibody (lower panel). IP: immunoprecipitation; IB: immunoblotting. (E) Upper part: Schematic representation of the deletion 5 soluble Hstalk-RFP. Lower part: alignment of the primary amino acid sequence of the CDV H-stalk “spacer” section and the derivative triple alanine-scan mutants. Alanines are highlighted in red. (G) Homology structural model of CDV-F and positions (in red) where the mAb αH-1347 epitope has been inserted. Lower part: primary amino acid sequence of the CDV H-stalk “spacer” section with the encompassed F-transferred mAb-1347 peptide (highlighted in red). (H) Reactivities of F-wt-tagFLAG or F-wt-tag1347 with the conformation-insensitive anti-FLAG, the F prefusion state-specific anti-F-Pre (4941) [ 61 , 63 ] or the anti-CDV-H 1347 mAbs, probed 24 hours post-transfection in receptor-negative Vero cells. After addition of the secondary antibody, MFI values were recorded by flow cytometry analyses. Means ± S.D. of data from three independent experiments in triplicate are shown.

    Techniques Used: Functional Assay, Immunoprecipitation, Derivative Assay, Transfection, Sequencing, Flow Cytometry, Cytometry

    Functional and biochemical characterization of stalk-elongated and shortened engineered H variants. (A) Upper part: schematic representation of the main functional domains of the full length CDV H-wt protein. Middle part: schematic representation of the H-elongated construct with the precise position of the insertion indicated. The 11 residues inserted are derived from the N-terminal stalk “contact” section 112–122 that putatively assume a helical fold with an 11-mer repeat. Lower part: schematic representation of the H-shortened version with the precise position of the deletion indicated. (B) Immunoblotting of standard and size-modulated H proteins. Antigenic materials ran in an 8% SDS-Page gel under reducing conditions were detected using a polyclonal anti-H antibody. (C) Syncytium formation assay. Fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H (or size-modulated mutants) and CDV F in the presence (+) of absence (-) of mAb αH-1347. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (D) Reactivity of H-wt and size-modulated H variants to mAbs αFLAG and αH-1347. Vero cells were transfected with the different H plasmids and, after addition of the secondary antibody, MFI values were recorded 24h post-transfection. (E) SLAM-binding activity of H mutants. Standard and H mutants were expressed in Vero cells. One day post-transfection, cell surface expression (CSE; recorded by anti-FLAG staining followed by flow cytometry analyses) and SLAM binding efficiency (calculated as described in the legend of Fig 1D , but without addition of mAbs before SLAM treatments) were determined. All values were normalized to H-wt. (F) Co-IPs, performed as described in the legend of Fig 4B , were obtained 24 hours post-transfection of cell extracts of Vero cells transfected with standard H or derivative mutants and F-wt-expressing vectors. (G) Semiquantitative assessment of F/H avidity of interactions. To quantify the avidities of F 1 -H interactions, the signals in each F 1 and H bands were quantified using the AIDA software package. The avidity of F 1 -H interactions is represented by the ratio of the amount of coimmunoprecipitated (coIP) F 1 over the product of F 1 in the cell lysates divided by the ratio of the amount of immunoprecipitated H over the product of H in the cell lysate ((coIP F 1 /TL F 1 )/(IP H/TL H)). Subsequently, all ratios were normalized to the ratio of the wild-type F-H interactions set to 100%. Averages represent at least two independent experiments. (H) Effect of SLAM treatment on mAb αH-1347’s H-binding activity. The assay and values were determined as described in the legend of Fig 4D (37°C). Means ± S.D. of data from two independent experiments performed in triplicates are shown.
    Figure Legend Snippet: Functional and biochemical characterization of stalk-elongated and shortened engineered H variants. (A) Upper part: schematic representation of the main functional domains of the full length CDV H-wt protein. Middle part: schematic representation of the H-elongated construct with the precise position of the insertion indicated. The 11 residues inserted are derived from the N-terminal stalk “contact” section 112–122 that putatively assume a helical fold with an 11-mer repeat. Lower part: schematic representation of the H-shortened version with the precise position of the deletion indicated. (B) Immunoblotting of standard and size-modulated H proteins. Antigenic materials ran in an 8% SDS-Page gel under reducing conditions were detected using a polyclonal anti-H antibody. (C) Syncytium formation assay. Fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H (or size-modulated mutants) and CDV F in the presence (+) of absence (-) of mAb αH-1347. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (D) Reactivity of H-wt and size-modulated H variants to mAbs αFLAG and αH-1347. Vero cells were transfected with the different H plasmids and, after addition of the secondary antibody, MFI values were recorded 24h post-transfection. (E) SLAM-binding activity of H mutants. Standard and H mutants were expressed in Vero cells. One day post-transfection, cell surface expression (CSE; recorded by anti-FLAG staining followed by flow cytometry analyses) and SLAM binding efficiency (calculated as described in the legend of Fig 1D , but without addition of mAbs before SLAM treatments) were determined. All values were normalized to H-wt. (F) Co-IPs, performed as described in the legend of Fig 4B , were obtained 24 hours post-transfection of cell extracts of Vero cells transfected with standard H or derivative mutants and F-wt-expressing vectors. (G) Semiquantitative assessment of F/H avidity of interactions. To quantify the avidities of F 1 -H interactions, the signals in each F 1 and H bands were quantified using the AIDA software package. The avidity of F 1 -H interactions is represented by the ratio of the amount of coimmunoprecipitated (coIP) F 1 over the product of F 1 in the cell lysates divided by the ratio of the amount of immunoprecipitated H over the product of H in the cell lysate ((coIP F 1 /TL F 1 )/(IP H/TL H)). Subsequently, all ratios were normalized to the ratio of the wild-type F-H interactions set to 100%. Averages represent at least two independent experiments. (H) Effect of SLAM treatment on mAb αH-1347’s H-binding activity. The assay and values were determined as described in the legend of Fig 4D (37°C). Means ± S.D. of data from two independent experiments performed in triplicates are shown.

    Techniques Used: Functional Assay, Construct, Derivative Assay, SDS Page, Tube Formation Assay, Activity Assay, Expressing, Transfection, Binding Assay, Staining, Flow Cytometry, Cytometry, Software, Co-Immunoprecipitation Assay, Immunoprecipitation

    Inhibition of CDV-mediated viral-cell and cell-cell fusion by the anti-CDV-H mAb-1347. (A) Syncytium formation assay. Cell-to-cell fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H-wt and CDV F-wt (A75/17 strain) in the presence of absence of mAb αH-1347. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (B) Quantitative fusion assay. Vero-cSLAM cells (target cells) were infected with MVA-T7 (MOI of 1). In parallel, a population of Vero cells (effector cells) was transfected with the F-wt and H-wt-expressing vectors and a plasmid containing the luciferase reporter gene under the control of the T7 promoter. Twelve hours after transfection, effector cells were mixed with target cells and seeded into fresh plates. After 2.5 h at 37°C, fusion was indirectly quantified by using a commercial luciferase-measuring kit. For each experiment, the value obtained for the standard F/H combination was set to 100%. (C) Virus neutralization assay. A total of 100 infectious units of recA75/17 red was incubated with the indicated dilution of antibody for 1 h at 37°C. The virus-antibody mixtures were then added to 3h on Vero cells, overlaid with agar-containing medium and further incubated for 72 h at 37°C. Cell entry efficiency was determined by counting the number of red fluorescent syncytia induced by recA75/17 red . (D) Effect of mAbs on H/SLAM binding efficiency. Vero cells were transfected with H-wt. Prior to treatment with soluble HA-tagged cSLAM molecules, mAbs were added as indicated, and SLAM-binding activity was calculated as the ratio of mean fluorescence intensities obtained with an anti-HA polyclonal Ab values (staining for sol. cSLAM) normalized to the levels obtained with the anti-FLAG mAb (staining for H). Values recorded for H-wt/cSLAM-binding efficiency in the absence of the mAb were set at 100%. Wt: wild type, α: monoclonal antibody, sSLAM: soluble version of cSLAM. Means ± S.D. of data from three independent experiments in triplicate are shown.
    Figure Legend Snippet: Inhibition of CDV-mediated viral-cell and cell-cell fusion by the anti-CDV-H mAb-1347. (A) Syncytium formation assay. Cell-to-cell fusion activity in Vero-cSLAM cells triggered by co-expression of CDV H-wt and CDV F-wt (A75/17 strain) in the presence of absence of mAb αH-1347. Representative fields of view of cell-cell fusion induced 24h post-transfection are shown. (B) Quantitative fusion assay. Vero-cSLAM cells (target cells) were infected with MVA-T7 (MOI of 1). In parallel, a population of Vero cells (effector cells) was transfected with the F-wt and H-wt-expressing vectors and a plasmid containing the luciferase reporter gene under the control of the T7 promoter. Twelve hours after transfection, effector cells were mixed with target cells and seeded into fresh plates. After 2.5 h at 37°C, fusion was indirectly quantified by using a commercial luciferase-measuring kit. For each experiment, the value obtained for the standard F/H combination was set to 100%. (C) Virus neutralization assay. A total of 100 infectious units of recA75/17 red was incubated with the indicated dilution of antibody for 1 h at 37°C. The virus-antibody mixtures were then added to 3h on Vero cells, overlaid with agar-containing medium and further incubated for 72 h at 37°C. Cell entry efficiency was determined by counting the number of red fluorescent syncytia induced by recA75/17 red . (D) Effect of mAbs on H/SLAM binding efficiency. Vero cells were transfected with H-wt. Prior to treatment with soluble HA-tagged cSLAM molecules, mAbs were added as indicated, and SLAM-binding activity was calculated as the ratio of mean fluorescence intensities obtained with an anti-HA polyclonal Ab values (staining for sol. cSLAM) normalized to the levels obtained with the anti-FLAG mAb (staining for H). Values recorded for H-wt/cSLAM-binding efficiency in the absence of the mAb were set at 100%. Wt: wild type, α: monoclonal antibody, sSLAM: soluble version of cSLAM. Means ± S.D. of data from three independent experiments in triplicate are shown.

    Techniques Used: Inhibition, Tube Formation Assay, Activity Assay, Expressing, Transfection, Single Vesicle Fusion Assay, Infection, Plasmid Preparation, Luciferase, Neutralization, Incubation, Binding Assay, Fluorescence, Staining

    2) Product Images from "Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment"

    Article Title: Apoptosis induction by Bid requires unconventional ubiquitination and degradation of its N-terminal fragment

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200707063

    Ubiquitin is partially reducible, hydrolyzable, and affected by combined mutation of S, T, and C residues. (A) HA tBid-N was coexpressed with FLAG ubiquitin in HeLa cells. Anti-HA immunoprecipitates were reduced (+) or not (−) as described in Materials and methods and reprecipitated with an anti-HA mAb. Reprecipitates were probed for Bid (anti-HA) and ubiquitin (anti-FLAG). Recombinant E2-25K as a positive control for cysteine ubiquitination was ubiquitinated in vitro, reduced or not, and probed with mAb to E2-25K or ubiquitin. The asterisk indicates free recombinant ubiquitin. IDV, integrated density values showing comparable tBid-N loading. (B) Alanine substitution mutations were generated in HA tBid-N TEV 30. All mutants listed in Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200707063/DC1 ) were tested for ubiquitination and part of the results are shown here. Wild-type and mutant proteins were expressed in HeLa cells together with FLAG ubiquitin, isolated with anti-HA mAb, and treated (+) or not (−) with TEV protease. Anti-HA immunoblotting was performed to assess Bid expression and cleavage and anti-FLAG immunoblotting was performed to examine ubiquitination. Arrows indicate positions of the ubiquitinated TEV cleavage fragment. (C) tBid-N TAP was coexpressed with HA-ubiquitin in HeLa cells. Cell lysates were treated at pH 7.4 or 12 as indicated in Materials and methods and tBid-N TAP was purified on the TAP tag. Calmodulin-bound protein was separated by SDS-PAGE and probed for Bid (anti-Bid) and ubiquitin (anti-HA). All experiments in this figure were performed three times with similar results. Molecular masses (kD) of marker proteins are indicated. Black lines indicate that intervening lanes have been spliced out.
    Figure Legend Snippet: Ubiquitin is partially reducible, hydrolyzable, and affected by combined mutation of S, T, and C residues. (A) HA tBid-N was coexpressed with FLAG ubiquitin in HeLa cells. Anti-HA immunoprecipitates were reduced (+) or not (−) as described in Materials and methods and reprecipitated with an anti-HA mAb. Reprecipitates were probed for Bid (anti-HA) and ubiquitin (anti-FLAG). Recombinant E2-25K as a positive control for cysteine ubiquitination was ubiquitinated in vitro, reduced or not, and probed with mAb to E2-25K or ubiquitin. The asterisk indicates free recombinant ubiquitin. IDV, integrated density values showing comparable tBid-N loading. (B) Alanine substitution mutations were generated in HA tBid-N TEV 30. All mutants listed in Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200707063/DC1 ) were tested for ubiquitination and part of the results are shown here. Wild-type and mutant proteins were expressed in HeLa cells together with FLAG ubiquitin, isolated with anti-HA mAb, and treated (+) or not (−) with TEV protease. Anti-HA immunoblotting was performed to assess Bid expression and cleavage and anti-FLAG immunoblotting was performed to examine ubiquitination. Arrows indicate positions of the ubiquitinated TEV cleavage fragment. (C) tBid-N TAP was coexpressed with HA-ubiquitin in HeLa cells. Cell lysates were treated at pH 7.4 or 12 as indicated in Materials and methods and tBid-N TAP was purified on the TAP tag. Calmodulin-bound protein was separated by SDS-PAGE and probed for Bid (anti-Bid) and ubiquitin (anti-HA). All experiments in this figure were performed three times with similar results. Molecular masses (kD) of marker proteins are indicated. Black lines indicate that intervening lanes have been spliced out.

    Techniques Used: Mutagenesis, Recombinant, Positive Control, In Vitro, Generated, Isolation, Expressing, Purification, SDS Page, Marker

    tBid-N is ubiquitinated in an unconventional manner. (A) HeLa cells were transfected to express HA tBid-N alone or in combination with FLAG-tagged ubiquitin. HA tBid-N was immunoprecipitated and probed with an anti-HA mAb to detect Bid and probed with anti-FLAG mAb to detect ubiquitin. Asterisks indicate heavy and light chains of anti-HA mAb. (B) MCF-7 cells expressing Myc Bid GFP were stimulated with TNF-α in the presence of MG132 for 8 h or not stimulated (control). Lysates were immunoprecipitated with an anti-GFP antibody and immunoprecipitated sequentially (seq) with an anti-Myc mAb, and immunoprecipitates (IP) were probed with an anti-Bid antibody and a P4D1 anti-ubiquitin mAb. (C) Assay to determine whether the N terminus is ubiquitinated. In the diagram, the tBid-N TAP proteins are assessed for ubiquitination. Cal–TEV–Prot A indicates the TAP tag with calmodulin- and IgG-binding sites separated by a cleavage site for TEV protease (arrows). HeLa cells were transfected to express HA-ubiquitin and TAP-tagged tBid-N. TAP-tagged tBid-N proteins were isolated with IgG beads, digested with TEV protease, and reisolated with calmodulin beads. Isolates were probed for ubiquitin with an anti-HA mAb and for tBid-N with an anti-Bid antibody. Note that in I, the Prot A sequence is still attached to the tBid-N TAP species and the anti-Bid antibody binds to it. Molecular masses (kD) of marker proteins are indicated.
    Figure Legend Snippet: tBid-N is ubiquitinated in an unconventional manner. (A) HeLa cells were transfected to express HA tBid-N alone or in combination with FLAG-tagged ubiquitin. HA tBid-N was immunoprecipitated and probed with an anti-HA mAb to detect Bid and probed with anti-FLAG mAb to detect ubiquitin. Asterisks indicate heavy and light chains of anti-HA mAb. (B) MCF-7 cells expressing Myc Bid GFP were stimulated with TNF-α in the presence of MG132 for 8 h or not stimulated (control). Lysates were immunoprecipitated with an anti-GFP antibody and immunoprecipitated sequentially (seq) with an anti-Myc mAb, and immunoprecipitates (IP) were probed with an anti-Bid antibody and a P4D1 anti-ubiquitin mAb. (C) Assay to determine whether the N terminus is ubiquitinated. In the diagram, the tBid-N TAP proteins are assessed for ubiquitination. Cal–TEV–Prot A indicates the TAP tag with calmodulin- and IgG-binding sites separated by a cleavage site for TEV protease (arrows). HeLa cells were transfected to express HA-ubiquitin and TAP-tagged tBid-N. TAP-tagged tBid-N proteins were isolated with IgG beads, digested with TEV protease, and reisolated with calmodulin beads. Isolates were probed for ubiquitin with an anti-HA mAb and for tBid-N with an anti-Bid antibody. Note that in I, the Prot A sequence is still attached to the tBid-N TAP species and the anti-Bid antibody binds to it. Molecular masses (kD) of marker proteins are indicated.

    Techniques Used: Transfection, Immunoprecipitation, Expressing, Binding Assay, Isolation, Sequencing, Marker

    Helix 1 is critical for tBid-N ubiquitination and degradation. (A) Wild-type (wt) tBid-N GFP and deletion (Δ) mutants lacking 7, 13, 15, 17, 23, or 33 N-terminal amino acids were expressed in HeLa cells, which were treated (+) or not (−) with CHX for 8 h. The tBid-N GFP proteins were detected in total lysates by immunoblotting with anti-Bid antibody. (B) GFP, wt, and Δ15 tBid-N GFP were expressed in HeLa cells together with FLAG-tagged ubiquitin. Total cell lysates (TCL) were probed with an anti-Bid antibody to assess tBid-N expression levels. Ubiquitinated protein species were isolated with an anti-FLAG mAb (IP) and probed with an anti-Bid antibody. Single asterisk indicates heavy and light chains of the anti-FLAG mAb; double asterisk indicates endogenous full-length Bid. (C) FLAG-tagged ubiquitin was coexpressed in HeLa cells with HA tBid-N versions with a TEV protease cleavage site at position 10, 30, or 43. HA tBid-N molecules were isolated, incubated with (+) or without (−) TEV protease, run on a gel, and probed by immunoblotting for Bid and ubiquitin (anti-FLAG). In the case of HA tBid-N TEV 10, the HA-tagged cleavage fragment repeatedly did not resolve on gel but the remaining undigested HA tBid-N TEV 10 and weakly ubiquitinated species of the cleavage fragment (arrows) are clearly visible. All experiments in this figure were performed three times with similar results. Molecular masses (kD) of marker proteins are indicated.
    Figure Legend Snippet: Helix 1 is critical for tBid-N ubiquitination and degradation. (A) Wild-type (wt) tBid-N GFP and deletion (Δ) mutants lacking 7, 13, 15, 17, 23, or 33 N-terminal amino acids were expressed in HeLa cells, which were treated (+) or not (−) with CHX for 8 h. The tBid-N GFP proteins were detected in total lysates by immunoblotting with anti-Bid antibody. (B) GFP, wt, and Δ15 tBid-N GFP were expressed in HeLa cells together with FLAG-tagged ubiquitin. Total cell lysates (TCL) were probed with an anti-Bid antibody to assess tBid-N expression levels. Ubiquitinated protein species were isolated with an anti-FLAG mAb (IP) and probed with an anti-Bid antibody. Single asterisk indicates heavy and light chains of the anti-FLAG mAb; double asterisk indicates endogenous full-length Bid. (C) FLAG-tagged ubiquitin was coexpressed in HeLa cells with HA tBid-N versions with a TEV protease cleavage site at position 10, 30, or 43. HA tBid-N molecules were isolated, incubated with (+) or without (−) TEV protease, run on a gel, and probed by immunoblotting for Bid and ubiquitin (anti-FLAG). In the case of HA tBid-N TEV 10, the HA-tagged cleavage fragment repeatedly did not resolve on gel but the remaining undigested HA tBid-N TEV 10 and weakly ubiquitinated species of the cleavage fragment (arrows) are clearly visible. All experiments in this figure were performed three times with similar results. Molecular masses (kD) of marker proteins are indicated.

    Techniques Used: Expressing, Isolation, Incubation, Marker

    3) Product Images from "Mutation of the Highly Conserved Ser-40 of the HIV-1 p6 Gag Protein to Phe Causes the Formation of a Hydrophobic Patch, Enhances Membrane Association, and Polyubiquitination of Gag"

    Article Title: Mutation of the Highly Conserved Ser-40 of the HIV-1 p6 Gag Protein to Phe Causes the Formation of a Hydrophobic Patch, Enhances Membrane Association, and Polyubiquitination of Gag

    Journal: Viruses

    doi: 10.3390/v6103738

    Overexpression of ALIX increases virus release but has no influence on Gag ubiquitination. ( A ) HeLa cells were co-transfected with HA-tagged ubiquitin, pΔR constructs as indicated, and either with, or without, FLAG-ALIX plasmid. Gag processing was inhibited by the addition of the PR-inhibitor Ritonavir. For detection of ubiquitinated Gag species, Gag was precipitated from whole cell lysates by immunoprecipitation using anti- HIV antibodies, and ubiquitin was detected by anti- HA staining. VLPs were pelleted from the supernatant and the amount of Gag released as VLPs was detected by anti- CA staining; ( B ) Evaluation of three independently performed experiments. Bars represent the arithmetic mean ± SD. The numbering is according to (A).
    Figure Legend Snippet: Overexpression of ALIX increases virus release but has no influence on Gag ubiquitination. ( A ) HeLa cells were co-transfected with HA-tagged ubiquitin, pΔR constructs as indicated, and either with, or without, FLAG-ALIX plasmid. Gag processing was inhibited by the addition of the PR-inhibitor Ritonavir. For detection of ubiquitinated Gag species, Gag was precipitated from whole cell lysates by immunoprecipitation using anti- HIV antibodies, and ubiquitin was detected by anti- HA staining. VLPs were pelleted from the supernatant and the amount of Gag released as VLPs was detected by anti- CA staining; ( B ) Evaluation of three independently performed experiments. Bars represent the arithmetic mean ± SD. The numbering is according to (A).

    Techniques Used: Over Expression, Transfection, Construct, Plasmid Preparation, Immunoprecipitation, Staining

    4) Product Images from "SUMOylation of the Lens Epithelium-derived Growth Factor/p75 attenuates its transcriptional activity on the Heat Shock Protein 27 promoter"

    Article Title: SUMOylation of the Lens Epithelium-derived Growth Factor/p75 attenuates its transcriptional activity on the Heat Shock Protein 27 promoter

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2010.03.063

    SUMOylation does not affect the chromatin binding properties of LEDGF. Chromatin-binding properties of LEDGF WT and SUMOylation-deficient mutants. (a) Chromatin-binding assay. LEDGF/p75-deficient HEK 293T cells co-expressing FLAG-tagged LEDGF/p75 WT and
    Figure Legend Snippet: SUMOylation does not affect the chromatin binding properties of LEDGF. Chromatin-binding properties of LEDGF WT and SUMOylation-deficient mutants. (a) Chromatin-binding assay. LEDGF/p75-deficient HEK 293T cells co-expressing FLAG-tagged LEDGF/p75 WT and

    Techniques Used: Binding Assay, Expressing

    LEDGF/p52 and LEDGF/p75 are SUMO-1 targets. (a) LEDGF/p75-deficient cells were transfected with FLAG-tagged LEDGF expression plasmids alone or together with the Dual S1/I/U plasmid encoding UBC9 and SUMO-1. Cell lysates were immunoblotted for LEDGF proteins
    Figure Legend Snippet: LEDGF/p52 and LEDGF/p75 are SUMO-1 targets. (a) LEDGF/p75-deficient cells were transfected with FLAG-tagged LEDGF expression plasmids alone or together with the Dual S1/I/U plasmid encoding UBC9 and SUMO-1. Cell lysates were immunoblotted for LEDGF proteins

    Techniques Used: Transfection, Expressing, Plasmid Preparation

    5) Product Images from "Src Homology Domain 2-containing Protein-tyrosine Phosphatase-1 (SHP-1) Binds and Dephosphorylates G?-interacting, Vesicle-associated Protein (GIV)/Girdin and Attenuates the GIV-Phosphatidylinositol 3-Kinase (PI3K)-Akt Signaling Pathway *"

    Article Title: Src Homology Domain 2-containing Protein-tyrosine Phosphatase-1 (SHP-1) Binds and Dephosphorylates G?-interacting, Vesicle-associated Protein (GIV)/Girdin and Attenuates the GIV-Phosphatidylinositol 3-Kinase (PI3K)-Akt Signaling Pathway *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.275685

    SHP-1 inhibits tyrosine phosphorylation of GIV by EGFR and Src. A , WT, but not the catalytically inactive CS mutant of SHP-1, dephosphorylates GIV after EGF stimulation. COS7 cells transfected with vector alone ( lane 1 ), GIV-FLAG alone ( lanes 2 and 3
    Figure Legend Snippet: SHP-1 inhibits tyrosine phosphorylation of GIV by EGFR and Src. A , WT, but not the catalytically inactive CS mutant of SHP-1, dephosphorylates GIV after EGF stimulation. COS7 cells transfected with vector alone ( lane 1 ), GIV-FLAG alone ( lanes 2 and 3

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation

    SHP-1 inhibits the GIV ability to enhance Akt phosphorylation. A and B , WT, but not the catalytically inactive CS mutant of SHP-1 inhibits GIV-dependent Akt phosphorylation. A , COS7 cells were transfected with empty vector ( lane 1 ), GIV-FLAG alone ( lane
    Figure Legend Snippet: SHP-1 inhibits the GIV ability to enhance Akt phosphorylation. A and B , WT, but not the catalytically inactive CS mutant of SHP-1 inhibits GIV-dependent Akt phosphorylation. A , COS7 cells were transfected with empty vector ( lane 1 ), GIV-FLAG alone ( lane

    Techniques Used: Mutagenesis, Transfection, Plasmid Preparation

    WT, but not the catalytically inactive SHP-1C453S ( CS ) mutant, inhibits tyrosine phosphorylation of GIV upon activation of LPA receptor. COS7 cells transfected with vector alone ( lane 1 ), GIV-FLAG alone ( lanes 2 and 3 ), GIV-FLAG and HA-SHP-1 WT ( lane
    Figure Legend Snippet: WT, but not the catalytically inactive SHP-1C453S ( CS ) mutant, inhibits tyrosine phosphorylation of GIV upon activation of LPA receptor. COS7 cells transfected with vector alone ( lane 1 ), GIV-FLAG alone ( lanes 2 and 3 ), GIV-FLAG and HA-SHP-1 WT ( lane

    Techniques Used: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation

    6) Product Images from "Variations of the UNC13D Gene in Patients with Autoimmune Lymphoproliferative Syndrome"

    Article Title: Variations of the UNC13D Gene in Patients with Autoimmune Lymphoproliferative Syndrome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068045

    Defective Fas function in the ALPS and DALD patients carrying the UNC13D variations. [A] Fas-induced cell death in T cells from the ALPS and DALD patients carrying the UNC13D variations. Activated T cells were treated with anti-Fas mAb and survival was assessed after 18 hours. The results are expressed as specific cell survival %. The dotted line indicates the upper limit of the normal range calculated as the 95 th percentile of data obtained from 200 healthy controls; two or more were run in each experiment as positive controls; each patient was evaluated at least twice with the same result. [B] Fas expression and caspase-8 activity in lysates of 293T cells transfected with the wild-type (WT) or mutated form of FAS (Pt.1: p.Gln273His, Pt.2: p.Glu261Lys); cells were lysed 24 hours after transfection. Upper panels : Western blot analysis of the transfected Fas performed using anti-FLAG and anti-β-actin antibodies . Lower panels : fluorimetric enzyme assay for caspase-8 activity. Data are relative to those displayed by mock-transfected cells and are expressed as the mean and SE of the results from 4 experiments performed in duplicate. *p
    Figure Legend Snippet: Defective Fas function in the ALPS and DALD patients carrying the UNC13D variations. [A] Fas-induced cell death in T cells from the ALPS and DALD patients carrying the UNC13D variations. Activated T cells were treated with anti-Fas mAb and survival was assessed after 18 hours. The results are expressed as specific cell survival %. The dotted line indicates the upper limit of the normal range calculated as the 95 th percentile of data obtained from 200 healthy controls; two or more were run in each experiment as positive controls; each patient was evaluated at least twice with the same result. [B] Fas expression and caspase-8 activity in lysates of 293T cells transfected with the wild-type (WT) or mutated form of FAS (Pt.1: p.Gln273His, Pt.2: p.Glu261Lys); cells were lysed 24 hours after transfection. Upper panels : Western blot analysis of the transfected Fas performed using anti-FLAG and anti-β-actin antibodies . Lower panels : fluorimetric enzyme assay for caspase-8 activity. Data are relative to those displayed by mock-transfected cells and are expressed as the mean and SE of the results from 4 experiments performed in duplicate. *p

    Techniques Used: Expressing, Activity Assay, Transfection, Western Blot, Enzymatic Assay

    7) Product Images from "The Structure Specific Recognition Protein 1 associates with Lens Epithelium-Derived Growth Factor proteins and modulates HIV-1 replication"

    Article Title: The Structure Specific Recognition Protein 1 associates with Lens Epithelium-Derived Growth Factor proteins and modulates HIV-1 replication

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2016.05.013

    Mapping the LEDGF regions implicated in SSRP1 binding (a) si1340/1428 cells were co-transfected with plasmids encoding Myc-SSRP1 and either LEDGF/p75-FLAG (lane 1), LEDGF/p52-FLAG (lane 2), C-terminal LEDGF/p75-FLAG (lane 3), or an empty expression plasmid (lane 4). Immunoprecipitation analyses were performed as previously described in figure legend 1a. (*) Marks degradation products of LEDGF/p52-FLAG. (b) Implication of PWWP domain in the interaction of LEDGF/p52 with SSRP1. Cell lysates were obtained from si1340/1428 cells co-transfected with plasmids expressing Myc-SSRP1 and either LEDGF/p52-FLAG (lane 1), LEDGF/p52ΔPWWP-FLAG (lane 2), or an empty plasmid (lane 3). Immunoprecipitations were performed as previously described. (c) si1340/1428 cells were co-transfected with plasmids expressing Myc-SSRP1 and PWWP-FLAG (lane 1) or an empty plasmid (lane 2). Immunoprecipitations were performed as previously described in figure legend 1a. Results in (c) are representative of two independent experiments. The line separating lanes 1 and 2 indicate that lanes of the gel in between contained irrelevant samples that were removed.
    Figure Legend Snippet: Mapping the LEDGF regions implicated in SSRP1 binding (a) si1340/1428 cells were co-transfected with plasmids encoding Myc-SSRP1 and either LEDGF/p75-FLAG (lane 1), LEDGF/p52-FLAG (lane 2), C-terminal LEDGF/p75-FLAG (lane 3), or an empty expression plasmid (lane 4). Immunoprecipitation analyses were performed as previously described in figure legend 1a. (*) Marks degradation products of LEDGF/p52-FLAG. (b) Implication of PWWP domain in the interaction of LEDGF/p52 with SSRP1. Cell lysates were obtained from si1340/1428 cells co-transfected with plasmids expressing Myc-SSRP1 and either LEDGF/p52-FLAG (lane 1), LEDGF/p52ΔPWWP-FLAG (lane 2), or an empty plasmid (lane 3). Immunoprecipitations were performed as previously described. (c) si1340/1428 cells were co-transfected with plasmids expressing Myc-SSRP1 and PWWP-FLAG (lane 1) or an empty plasmid (lane 2). Immunoprecipitations were performed as previously described in figure legend 1a. Results in (c) are representative of two independent experiments. The line separating lanes 1 and 2 indicate that lanes of the gel in between contained irrelevant samples that were removed.

    Techniques Used: Binding Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

    Immunoprecipitation analyses of the interaction of SSRP1 mutants with LEDGF/p75 (a) HEK293T cells were co-transfected with plasmids expressing: FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1 WT (lane 1), FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1ΔHMG (lane 2), or Myc-tagged SSRP1 WT and an empty plasmid (lane 3). Results in (a) are representative of three independent experiments. The vertical lines separating lanes 1 and 2 indicate that samples were in the same gel but not in adjacent positions. (*) Denotes residual SSRP1 resulting from partial antibody stripping. (b) HEK293T cells were co-transfected with plasmids encoding LEDGF/p75-FLAG and SSRP1 WT-Myc (lane 1) or HMG-Myc (lane 2). Lane 3 was co-transfected with SSRP1 WT-Myc and an empty plasmid. S1 and S2 fractions were obtained from the transfected cells and mixed. Mixed fractions were immunoprecipitated as described in figure legend 1a. Results are representative of two independent experiments.
    Figure Legend Snippet: Immunoprecipitation analyses of the interaction of SSRP1 mutants with LEDGF/p75 (a) HEK293T cells were co-transfected with plasmids expressing: FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1 WT (lane 1), FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1ΔHMG (lane 2), or Myc-tagged SSRP1 WT and an empty plasmid (lane 3). Results in (a) are representative of three independent experiments. The vertical lines separating lanes 1 and 2 indicate that samples were in the same gel but not in adjacent positions. (*) Denotes residual SSRP1 resulting from partial antibody stripping. (b) HEK293T cells were co-transfected with plasmids encoding LEDGF/p75-FLAG and SSRP1 WT-Myc (lane 1) or HMG-Myc (lane 2). Lane 3 was co-transfected with SSRP1 WT-Myc and an empty plasmid. S1 and S2 fractions were obtained from the transfected cells and mixed. Mixed fractions were immunoprecipitated as described in figure legend 1a. Results are representative of two independent experiments.

    Techniques Used: Immunoprecipitation, Transfection, Expressing, Plasmid Preparation, Stripping Membranes

    Interaction of LEDGF/p75 with the FACT complex (a) Chromatin-bound proteins were isolated from T L3 and T L3 LEDGF/p75 WT cells by DNase treatment and FLAG-tagged LEDGF/p75 was immunoprecipitated from this subcellular fraction with an anti-FLAG mAb antibody. The presence of the FACT complex (hSpt16 and SSRP1) was evaluated in the immunoprecipitated proteins by immunoblotting with specific antibodies. (b) Quantitative confocal microscopy co-localization of LEDGF/p75 with SSRP1. Panels 1 and 2 are controls and represent the co-localization of HIV integrase with LEDGF/p75 mutant lacking the integrase-binding domain (ΔIBD) or LEDGF/p75 wild type (WT). LEDGF/p75-deficient HEK293T cells stably expressing eGFP-tagged HIV-1 integrase were transiently transfected with LEDGF/p75ΔIBD or WT. LEDGF/p75 proteins were detected with an anti-LEDGF antibody. The lower panels represent the co-localization of endogenous LEDGF/p75 with endogenous SSRP1 or Pol II in HeLa cells after immunostaining with specific antibodies. (c) Quantification of co-localization data in (b). Standard deviations indicated co-localization values found in ten different cells randomly selected from a field representative of ten different random areas of the microscope slide. (d) HEK293T cells were co-transfected with plasmids expressing: FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1 WT (lane 1), FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1ΔNTD (lane 2), or Myc-tagged SSRP1 WT and an empty plasmid (lane 3). Samples were analyzed in the same gel, the line dividing lanes 2 and 3 indicate that lanes in between were removed for comparison in this figure. In 1d, (*) indicates degradation products of SSRP1
    Figure Legend Snippet: Interaction of LEDGF/p75 with the FACT complex (a) Chromatin-bound proteins were isolated from T L3 and T L3 LEDGF/p75 WT cells by DNase treatment and FLAG-tagged LEDGF/p75 was immunoprecipitated from this subcellular fraction with an anti-FLAG mAb antibody. The presence of the FACT complex (hSpt16 and SSRP1) was evaluated in the immunoprecipitated proteins by immunoblotting with specific antibodies. (b) Quantitative confocal microscopy co-localization of LEDGF/p75 with SSRP1. Panels 1 and 2 are controls and represent the co-localization of HIV integrase with LEDGF/p75 mutant lacking the integrase-binding domain (ΔIBD) or LEDGF/p75 wild type (WT). LEDGF/p75-deficient HEK293T cells stably expressing eGFP-tagged HIV-1 integrase were transiently transfected with LEDGF/p75ΔIBD or WT. LEDGF/p75 proteins were detected with an anti-LEDGF antibody. The lower panels represent the co-localization of endogenous LEDGF/p75 with endogenous SSRP1 or Pol II in HeLa cells after immunostaining with specific antibodies. (c) Quantification of co-localization data in (b). Standard deviations indicated co-localization values found in ten different cells randomly selected from a field representative of ten different random areas of the microscope slide. (d) HEK293T cells were co-transfected with plasmids expressing: FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1 WT (lane 1), FLAG-tagged LEDGF/p75 and Myc-tagged SSRP1ΔNTD (lane 2), or Myc-tagged SSRP1 WT and an empty plasmid (lane 3). Samples were analyzed in the same gel, the line dividing lanes 2 and 3 indicate that lanes in between were removed for comparison in this figure. In 1d, (*) indicates degradation products of SSRP1

    Techniques Used: Isolation, Immunoprecipitation, Confocal Microscopy, Mutagenesis, Binding Assay, Stable Transfection, Expressing, Transfection, Immunostaining, Microscopy, Plasmid Preparation

    8) Product Images from "Formation of a WIP-, WASp-, actin-, and myosin IIA-containing multiprotein complex in activated NK cells and its alteration by KIR inhibitory signaling"

    Article Title: Formation of a WIP-, WASp-, actin-, and myosin IIA-containing multiprotein complex in activated NK cells and its alteration by KIR inhibitory signaling

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200509076

    Actin and myosin IIA interaction with WIP is independent of WASp. YTS/KIR2DL1 cells were transfected with FLAG-WIP or FLAG-WIPΔ460-503, which is a mutant protein lacking the WASp binding domain. Transfected cells were mixed with 721.221 cells and incubated for the indicated times at 37°C. Cell lysates were immunoprecipitated with anti-FLAG mAb. Immunoprecipitated proteins were immunoblotted with anti–myosin IIA, anti-FLAG, anti-WASp, or anti-actin antibodies. The molecular masses of the proteins according to their position relative to molecular mass markers are shown in parentheses. The absence of WASp does not affect recruitment of either actin or myosin IIA to the complex.
    Figure Legend Snippet: Actin and myosin IIA interaction with WIP is independent of WASp. YTS/KIR2DL1 cells were transfected with FLAG-WIP or FLAG-WIPΔ460-503, which is a mutant protein lacking the WASp binding domain. Transfected cells were mixed with 721.221 cells and incubated for the indicated times at 37°C. Cell lysates were immunoprecipitated with anti-FLAG mAb. Immunoprecipitated proteins were immunoblotted with anti–myosin IIA, anti-FLAG, anti-WASp, or anti-actin antibodies. The molecular masses of the proteins according to their position relative to molecular mass markers are shown in parentheses. The absence of WASp does not affect recruitment of either actin or myosin IIA to the complex.

    Techniques Used: Transfection, Mutagenesis, Binding Assay, Incubation, Immunoprecipitation

    WIP, WASp, and myosin IIA polarize to cell–cell contact site after NK cell activation, but not after inhibition. YTS/KIR2DL1/FLAG-WIP cells, unconjugated or conjugated by mixing for 10 min at 37°C with target cells (either 721.221 [cytolytic interaction] or 721.221/Cw6 [noncytolytic interaction]), were stained with anti-WASp mAb followed by Alexa Fluor 647–conjugated goat anti–mouse (green), anti–myosin IIA followed by Alexa Fluor 405–conjugated goat anti–rabbit (blue), and Cy3-conjugated anti-FLAG mAb (red). NK cells were identified by GFP fluorescence and anti-FLAG staining. (A) Images of FLAG-WIP, WASp, and myosin IIA localization in resting cells (top), cytolytic (middle), and noncytolytic (bottom) conjugates. (B) The percentages of FLAG-WIP, WASp, and myosin IIA polarization toward cell–cell contact site in cytolytic (left) or noncytolytic (right) conjugates. Percentages of proteins polarized and found at the contact site (total polarization), or proteins found exclusively at the cell–cell contact site (synaptic accumulation), and percentages of proteins not polarized to the contact site (no polarization) ± SD are shown. The values were determined by evaluation of 150–200 conjugates in 3–4 separate experiments. n = 200 for WIP and WASp; n = 150 for myosin IIA.
    Figure Legend Snippet: WIP, WASp, and myosin IIA polarize to cell–cell contact site after NK cell activation, but not after inhibition. YTS/KIR2DL1/FLAG-WIP cells, unconjugated or conjugated by mixing for 10 min at 37°C with target cells (either 721.221 [cytolytic interaction] or 721.221/Cw6 [noncytolytic interaction]), were stained with anti-WASp mAb followed by Alexa Fluor 647–conjugated goat anti–mouse (green), anti–myosin IIA followed by Alexa Fluor 405–conjugated goat anti–rabbit (blue), and Cy3-conjugated anti-FLAG mAb (red). NK cells were identified by GFP fluorescence and anti-FLAG staining. (A) Images of FLAG-WIP, WASp, and myosin IIA localization in resting cells (top), cytolytic (middle), and noncytolytic (bottom) conjugates. (B) The percentages of FLAG-WIP, WASp, and myosin IIA polarization toward cell–cell contact site in cytolytic (left) or noncytolytic (right) conjugates. Percentages of proteins polarized and found at the contact site (total polarization), or proteins found exclusively at the cell–cell contact site (synaptic accumulation), and percentages of proteins not polarized to the contact site (no polarization) ± SD are shown. The values were determined by evaluation of 150–200 conjugates in 3–4 separate experiments. n = 200 for WIP and WASp; n = 150 for myosin IIA.

    Techniques Used: Activation Assay, Inhibition, Staining, Fluorescence

    A multiprotein complex formed during NK cell activation is altered by inhibitory signaling. (A) YTS/KIR2DL1/FLAG-WIP cells were mixed with 721.221 or 721.221/Cw6 cells, immediately transferred to 37°C, and incubated for the indicated times. Cell lysates were immunoprecipitated with anti-FLAG mAb. Immunoprecipitated proteins were resolved on a 4–12% NuPage gel and stained with silver. The proteins recruited to the complex (marked by numbered asterisks) were subsequently analyzed by MS. (B) An example of vacuum matrix–assisted laser disorption ionization MS analysis of the band corresponding to the first asterisk (*1) in the silver-stained gel in A. The lettered peaks obtained were identified as matching myosin IIA heavy chain and are overlaid on a schematic of the 1,960–amino acid residue molecule (bottom). The width of the individual box is representative of the size of the given peptide. Boxes shown in dark gray were further positively identified by MS peptide sequence. (C) Western blotting analysis of the anti-FLAG–immunoprecipitated proteins shown in A. The same PVDF membrane was sequentially stripped and probed with anti–myosin IIA, anti-FLAG, anti-WASp, anti-actin, and anti–myosin light chain antibodies. The molecular masses of the proteins according to their position, relative to molecular mass markers, are shown in parentheses. (D) A KIR2DL1 mutant, lacking the ITIM motifs, is unable to affect the formation of the complex. YTS/KIR2DL1*250/FLAG-WIP cells were mixed with 721.221/Cw6 cells, immediately transferred to 37°C, and incubated for the indicated times. Cell lysates were immunoprecipitated with anti-FLAG mAb. Immunoprecipitated proteins were visualized by immunoblotting with anti–myosin IIA, anti-FLAG, anti-WASp, anti-actin, and anti–myosin regulatory light chain antibodies. (E) Effects of actin inhibitors on the interaction of WIP and actin. YTS/KIR2DL1/FLAG-WIP cells were pretreated with DMSO (controls) or with the following actin inhibitors: latrunculin A (LatA), latrunculin B (LatB), jasplakinolide (Jasp), or swinholide A (SwA) at the indicated concentrations for 30 min at 37°C, followed by mixing with 721.221 target cells (except for the negative control) for 10 min at 37°C. Cell lysates were immunoprecipitated with anti-FLAG mAb and immunoblotted with anti-FLAG and anti-actin antibodies. pos. contr., cells pretreated with 0.1% DMSO and activated by mixing with 721.221 target cells (positive control); neg. contr., resting cells treated with 0.1% DMSO (negative control).
    Figure Legend Snippet: A multiprotein complex formed during NK cell activation is altered by inhibitory signaling. (A) YTS/KIR2DL1/FLAG-WIP cells were mixed with 721.221 or 721.221/Cw6 cells, immediately transferred to 37°C, and incubated for the indicated times. Cell lysates were immunoprecipitated with anti-FLAG mAb. Immunoprecipitated proteins were resolved on a 4–12% NuPage gel and stained with silver. The proteins recruited to the complex (marked by numbered asterisks) were subsequently analyzed by MS. (B) An example of vacuum matrix–assisted laser disorption ionization MS analysis of the band corresponding to the first asterisk (*1) in the silver-stained gel in A. The lettered peaks obtained were identified as matching myosin IIA heavy chain and are overlaid on a schematic of the 1,960–amino acid residue molecule (bottom). The width of the individual box is representative of the size of the given peptide. Boxes shown in dark gray were further positively identified by MS peptide sequence. (C) Western blotting analysis of the anti-FLAG–immunoprecipitated proteins shown in A. The same PVDF membrane was sequentially stripped and probed with anti–myosin IIA, anti-FLAG, anti-WASp, anti-actin, and anti–myosin light chain antibodies. The molecular masses of the proteins according to their position, relative to molecular mass markers, are shown in parentheses. (D) A KIR2DL1 mutant, lacking the ITIM motifs, is unable to affect the formation of the complex. YTS/KIR2DL1*250/FLAG-WIP cells were mixed with 721.221/Cw6 cells, immediately transferred to 37°C, and incubated for the indicated times. Cell lysates were immunoprecipitated with anti-FLAG mAb. Immunoprecipitated proteins were visualized by immunoblotting with anti–myosin IIA, anti-FLAG, anti-WASp, anti-actin, and anti–myosin regulatory light chain antibodies. (E) Effects of actin inhibitors on the interaction of WIP and actin. YTS/KIR2DL1/FLAG-WIP cells were pretreated with DMSO (controls) or with the following actin inhibitors: latrunculin A (LatA), latrunculin B (LatB), jasplakinolide (Jasp), or swinholide A (SwA) at the indicated concentrations for 30 min at 37°C, followed by mixing with 721.221 target cells (except for the negative control) for 10 min at 37°C. Cell lysates were immunoprecipitated with anti-FLAG mAb and immunoblotted with anti-FLAG and anti-actin antibodies. pos. contr., cells pretreated with 0.1% DMSO and activated by mixing with 721.221 target cells (positive control); neg. contr., resting cells treated with 0.1% DMSO (negative control).

    Techniques Used: Activation Assay, Incubation, Immunoprecipitation, Staining, Mass Spectrometry, Sequencing, Western Blot, Mutagenesis, Negative Control, Positive Control

    WIP phosphorylation increases upon NK cell activation and is mediated by PKCθ. (A) 32 P-labeled YTS/KIR2DL1/FLAG-WIP cells were mixed with target cells, either 721.221 or 721.221/Cw6, transferred to 37°C, and incubated for the indicated times. Cell lysates were immunoprecipitated with anti-FLAG mAb. Immunoprecipitated proteins were visualized by autoradiography and then immunoblotted with anti-FLAG antibody, followed by anti-WASp to visualize WIP and WASp, respectively. (B) Quantitation of WIP phosphorylation showing increase of WIP phosphorylation with maximum at 10 min of NK cell activation. The increase in phosphorylation of WIP was assessed by densitometry, which was normalized to phosphorylation observed at time 0 min and expressed as arbitrary units. (C) 32 P-labeled cells were pretreated with 0.1% DMSO (control) or with the following kinase inhibitors: bisindolylmaleimide I (Bisi.), PKCα and PKCβ pseudosubstrate inhibitor (αβ inh ), PKCθ pseudosubstrate inhibitor (θ inh ), casein kinase II inhibitor (CK2 inh ), Src family kinase inhibitor (PP2), blebbistatin (BL), or ML-7 at the indicated concentrations for 30 min at 37°C, followed by mixing with 721.221 cells for 10 min at 37°C. Cell lysates were immunoprecipitated with anti-FLAG mAb and treated similar to the cells in A, but using anti-FLAG, anti–myosin IIA, anti-actin, and anti-WASp antibodies sequentially for immunoblotting. The molecular masses of the proteins according to their position relative to molecular mass markers are shown in parentheses.
    Figure Legend Snippet: WIP phosphorylation increases upon NK cell activation and is mediated by PKCθ. (A) 32 P-labeled YTS/KIR2DL1/FLAG-WIP cells were mixed with target cells, either 721.221 or 721.221/Cw6, transferred to 37°C, and incubated for the indicated times. Cell lysates were immunoprecipitated with anti-FLAG mAb. Immunoprecipitated proteins were visualized by autoradiography and then immunoblotted with anti-FLAG antibody, followed by anti-WASp to visualize WIP and WASp, respectively. (B) Quantitation of WIP phosphorylation showing increase of WIP phosphorylation with maximum at 10 min of NK cell activation. The increase in phosphorylation of WIP was assessed by densitometry, which was normalized to phosphorylation observed at time 0 min and expressed as arbitrary units. (C) 32 P-labeled cells were pretreated with 0.1% DMSO (control) or with the following kinase inhibitors: bisindolylmaleimide I (Bisi.), PKCα and PKCβ pseudosubstrate inhibitor (αβ inh ), PKCθ pseudosubstrate inhibitor (θ inh ), casein kinase II inhibitor (CK2 inh ), Src family kinase inhibitor (PP2), blebbistatin (BL), or ML-7 at the indicated concentrations for 30 min at 37°C, followed by mixing with 721.221 cells for 10 min at 37°C. Cell lysates were immunoprecipitated with anti-FLAG mAb and treated similar to the cells in A, but using anti-FLAG, anti–myosin IIA, anti-actin, and anti-WASp antibodies sequentially for immunoblotting. The molecular masses of the proteins according to their position relative to molecular mass markers are shown in parentheses.

    Techniques Used: Activation Assay, Labeling, Incubation, Immunoprecipitation, Autoradiography, Quantitation Assay

    9) Product Images from "Secreted tyrosine sulfated-eIF5A mediates oxidative stress-induced apoptosis"

    Article Title: Secreted tyrosine sulfated-eIF5A mediates oxidative stress-induced apoptosis

    Journal: Scientific Reports

    doi: 10.1038/srep13737

    Isolation and identification of an apoptosis-inducing protein secreted from cardiac myocytes in response to hypoxia/reoxygenation. ( a ) Chromatofocusing of CCP (left panel) and RCP (Right panel) from cultured cardiac myocytes. The blue and red bars indicate the ERK-inducing effect of each fraction on the cultured cardiac myocytes. ( b ) 2-D gel electrophoresis of the ERK-activating fractions (fractions 49–52) from CCP (left panel) or RCP (right panel) followed by silver staining. The arrows indicate protein spots in the RCP (left panel) that were not detected or were very weakly detected (2′) in the CCP (left panel). ( c ) Western blot analysis of cytosolic re-eIF5A from untreated transfected cells (left panel) and re-eIF5A from the RCP (right panel); the membrane was probed with an anti-FLAG mAb and was developed via chemiluminescence using alkaline phosphatase. ( d ) Magnification of the spots shown in c (cytosolic re-eIF5A [upper panel] and re-eIF5A from RCP [lower panel]). The arrows indicate the unhypusinated (A and A’), deoxyhypusinated (B and B’), and hypusinated forms of eIF5A (C and C’). ( e ) The hypusinated/unhypusinated ratio (mean ± s.e.m.) for cytosolic and secreted re-eIF5A (n = 4 for each, * P = 0.0209) (Mann-Whitney U -test).
    Figure Legend Snippet: Isolation and identification of an apoptosis-inducing protein secreted from cardiac myocytes in response to hypoxia/reoxygenation. ( a ) Chromatofocusing of CCP (left panel) and RCP (Right panel) from cultured cardiac myocytes. The blue and red bars indicate the ERK-inducing effect of each fraction on the cultured cardiac myocytes. ( b ) 2-D gel electrophoresis of the ERK-activating fractions (fractions 49–52) from CCP (left panel) or RCP (right panel) followed by silver staining. The arrows indicate protein spots in the RCP (left panel) that were not detected or were very weakly detected (2′) in the CCP (left panel). ( c ) Western blot analysis of cytosolic re-eIF5A from untreated transfected cells (left panel) and re-eIF5A from the RCP (right panel); the membrane was probed with an anti-FLAG mAb and was developed via chemiluminescence using alkaline phosphatase. ( d ) Magnification of the spots shown in c (cytosolic re-eIF5A [upper panel] and re-eIF5A from RCP [lower panel]). The arrows indicate the unhypusinated (A and A’), deoxyhypusinated (B and B’), and hypusinated forms of eIF5A (C and C’). ( e ) The hypusinated/unhypusinated ratio (mean ± s.e.m.) for cytosolic and secreted re-eIF5A (n = 4 for each, * P = 0.0209) (Mann-Whitney U -test).

    Techniques Used: Isolation, Cell Culture, Nucleic Acid Electrophoresis, Silver Staining, Western Blot, Transfection, MANN-WHITNEY

    10) Product Images from "CD1d, a Sentinel Molecule Bridging Innate and Adaptive Immunity, Is Downregulated by the Human Papillomavirus (HPV) E5 Protein: a Possible Mechanism for Immune Evasion by HPV ▿"

    Article Title: CD1d, a Sentinel Molecule Bridging Innate and Adaptive Immunity, Is Downregulated by the Human Papillomavirus (HPV) E5 Protein: a Possible Mechanism for Immune Evasion by HPV ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01053-10

    CD1d and calnexin have direct interactions and colocalize in the perinuclear area in the presence of HPV E5. (A) Protein lysates from C33A/CD1d-empty, C33A/CD1d-6E5, and C33A/CD1d-16E5 cells were immunoprecipitated with an anti-FLAG MAb. Immunoprecipitants
    Figure Legend Snippet: CD1d and calnexin have direct interactions and colocalize in the perinuclear area in the presence of HPV E5. (A) Protein lysates from C33A/CD1d-empty, C33A/CD1d-6E5, and C33A/CD1d-16E5 cells were immunoprecipitated with an anti-FLAG MAb. Immunoprecipitants

    Techniques Used: Immunoprecipitation

    11) Product Images from "The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy"

    Article Title: The Inhibitory Core of the Myostatin Prodomain: Its Interaction with Both Type I and II Membrane Receptors, and Potential to Treat Muscle Atrophy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0133713

    Interaction of the inhibitory core of myostatin with its ligand and receptors. Co-localization ( Upper ) and co-immunoprecipitation ( Lower ) of the inhibitory core (IC) of the myostatin prodomain and its ligand ( A ), its type I receptors (ALK4 and ALK5, B ), and its type II receptors (ActRIIA and ActRIIB, C ) in COS-7 embryonic kidney cells expressing FLAG-tagged IC and V5- or HA-tagged ligand or receptors. Scale bar, 20 μm. Whole cell extracts (WCE) were immunoprecipitated with anti-FLAG, anti-V5, or anti-HA agarose and then immunoblotted using anti-FLAG, anti-V5, or anti-HA antibodies, respectively.
    Figure Legend Snippet: Interaction of the inhibitory core of myostatin with its ligand and receptors. Co-localization ( Upper ) and co-immunoprecipitation ( Lower ) of the inhibitory core (IC) of the myostatin prodomain and its ligand ( A ), its type I receptors (ALK4 and ALK5, B ), and its type II receptors (ActRIIA and ActRIIB, C ) in COS-7 embryonic kidney cells expressing FLAG-tagged IC and V5- or HA-tagged ligand or receptors. Scale bar, 20 μm. Whole cell extracts (WCE) were immunoprecipitated with anti-FLAG, anti-V5, or anti-HA agarose and then immunoblotted using anti-FLAG, anti-V5, or anti-HA antibodies, respectively.

    Techniques Used: Immunoprecipitation, Expressing

    12) Product Images from "Plexin B3 promotes neurite outgrowth, interacts homophilically, and interacts with Rin"

    Article Title: Plexin B3 promotes neurite outgrowth, interacts homophilically, and interacts with Rin

    Journal: BMC Neuroscience

    doi: 10.1186/1471-2202-6-53

    Co-immunoprecipitation (IP) experiments showing homophilic interaction of human and murine B3 and no interaction of B3 with plexins A1, B1 or B2 . Total lysates and IPs were analyzed by Western blot (WB) using antibodies as indicated in the figures. IP was performed with pAbB3-B against human B3 and shows interaction between mouse and human B3 and no interaction between B3 and human plexins A1, B1 and B2. (A) Cells were co-transfected with pSecTag2B/B3 encoding myc-tagged human full-length B3 and pcDNA3.1/mB3 encoding V5-tagged mouse B3 lacking most of its intracellular part. Cells transfected with pcDNA3.1/mB3 and pSecTag2B vector without insert served as negative control. (B) COS-7 cells were co-transfected with pIRES/B3 encoding non-tagged full-length human B3 and pcDNAVSV/A1 encoding VSV-tagged full-length human plexin A1. Cells co-transfected with pcDNAVSV/A1 and pIRES vector without insert served as negative control. (C) COS-7 cells were co-transfected with pIRES/B3 and pcDNAVSV/B1 encoding VSV-tagged full-length human plexin B1. Cells co-transfected with pcDNAVSV/B1 and pIRES vector without insert served as negative control. (D) COS-7 cells were co-transfected with pIRES/B3 and pFLAG/B2 encoding FLAG-tagged full-length human plexin B2. Cells co-transfected with pFLAG/B2 and pIRES vector without insert served as negative control.
    Figure Legend Snippet: Co-immunoprecipitation (IP) experiments showing homophilic interaction of human and murine B3 and no interaction of B3 with plexins A1, B1 or B2 . Total lysates and IPs were analyzed by Western blot (WB) using antibodies as indicated in the figures. IP was performed with pAbB3-B against human B3 and shows interaction between mouse and human B3 and no interaction between B3 and human plexins A1, B1 and B2. (A) Cells were co-transfected with pSecTag2B/B3 encoding myc-tagged human full-length B3 and pcDNA3.1/mB3 encoding V5-tagged mouse B3 lacking most of its intracellular part. Cells transfected with pcDNA3.1/mB3 and pSecTag2B vector without insert served as negative control. (B) COS-7 cells were co-transfected with pIRES/B3 encoding non-tagged full-length human B3 and pcDNAVSV/A1 encoding VSV-tagged full-length human plexin A1. Cells co-transfected with pcDNAVSV/A1 and pIRES vector without insert served as negative control. (C) COS-7 cells were co-transfected with pIRES/B3 and pcDNAVSV/B1 encoding VSV-tagged full-length human plexin B1. Cells co-transfected with pcDNAVSV/B1 and pIRES vector without insert served as negative control. (D) COS-7 cells were co-transfected with pIRES/B3 and pFLAG/B2 encoding FLAG-tagged full-length human plexin B2. Cells co-transfected with pFLAG/B2 and pIRES vector without insert served as negative control.

    Techniques Used: Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Negative Control

    13) Product Images from "The Golgi Localization of GOLPH2 (GP73/GOLM1) Is Determined by the Transmembrane and Cytoplamic Sequences"

    Article Title: The Golgi Localization of GOLPH2 (GP73/GOLM1) Is Determined by the Transmembrane and Cytoplamic Sequences

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0028207

    The TMD and cytoplasmic tail of GOLPH2 determine its Golgi localization. A. Subcellular localization of GOLPH2 truncation proteins in HeLa cells. Recombinant proteins transiently expressed in HeLa cells were stained with anti-FLAG mAb, which emits red fluorescence. Subcellular localization was imaged using a confocal microscope. β-1,4-galactosyltransferase (GalT) fused with cyan fluorescent protein was cotransfected as a Golgi localization marker (shown as a green pseudocolor). Merged images show the colocalization of GOLPH2 truncation proteins and GalT. Bars, 10 µm. B. The TMD and cytoplasmic tail of GOLPH2 were sufficient for Golgi localization. HeLa cells were transfected with GOLPH2-Δ(III–IV–V), GOLPH2-Δ(I–III–IV–V), and pEGFP. About 24 h after transfection, cells were treated for 4 h with cycloheximide (100 µg/mL) to inhibit protein synthesis prior to fixation and permeabilization. Subcellular localization was imaged using a confocal microscope. Endogenous GOLPH2 was probed using anti-GOLPH2 mAb as a Golgi localization marker (red fluorescence). Merged images were analyzed using the Leica confocal software. Bars, 10 µm. C. Deletion of region I resulted in ER localization. GOLPH2-Δ(III–IV–V), GOLPH2-Δ(I–III–IV–V), GOLPH2-ΔI, and pEGFP were transfected into HeLa cells. Subcellular localization was imaged using a confocal microscope. Calreticulin was probed as an ER marker protein (red fluorescence).
    Figure Legend Snippet: The TMD and cytoplasmic tail of GOLPH2 determine its Golgi localization. A. Subcellular localization of GOLPH2 truncation proteins in HeLa cells. Recombinant proteins transiently expressed in HeLa cells were stained with anti-FLAG mAb, which emits red fluorescence. Subcellular localization was imaged using a confocal microscope. β-1,4-galactosyltransferase (GalT) fused with cyan fluorescent protein was cotransfected as a Golgi localization marker (shown as a green pseudocolor). Merged images show the colocalization of GOLPH2 truncation proteins and GalT. Bars, 10 µm. B. The TMD and cytoplasmic tail of GOLPH2 were sufficient for Golgi localization. HeLa cells were transfected with GOLPH2-Δ(III–IV–V), GOLPH2-Δ(I–III–IV–V), and pEGFP. About 24 h after transfection, cells were treated for 4 h with cycloheximide (100 µg/mL) to inhibit protein synthesis prior to fixation and permeabilization. Subcellular localization was imaged using a confocal microscope. Endogenous GOLPH2 was probed using anti-GOLPH2 mAb as a Golgi localization marker (red fluorescence). Merged images were analyzed using the Leica confocal software. Bars, 10 µm. C. Deletion of region I resulted in ER localization. GOLPH2-Δ(III–IV–V), GOLPH2-Δ(I–III–IV–V), GOLPH2-ΔI, and pEGFP were transfected into HeLa cells. Subcellular localization was imaged using a confocal microscope. Calreticulin was probed as an ER marker protein (red fluorescence).

    Techniques Used: Recombinant, Staining, Fluorescence, Microscopy, Marker, Transfection, Software

    14) Product Images from "Neutrophils and the S100A9 protein critically regulate granuloma formation"

    Article Title: Neutrophils and the S100A9 protein critically regulate granuloma formation

    Journal: Blood Advances

    doi: 10.1182/bloodadvances.2016000497

    Identification of the G213-reactive protein as S100A9 . (A) Tissue lysates derived from the lungs of untreated (left lane) and BCG-challenged (right lane) guinea pigs were resolved on SDS-PAGE, and western blotting was conducted with G213 (upper panel) and Ab to β-actin (lower panel). The 15-kDa species specifically recognized by G213 is indicated with an asterisk. The 30-kDa bands observed on both lanes may represent nonspecific signals. (B) Tissue lysates derived from the bone marrow, spleen, lymph nodes, and thymus of an untreated guinea pig were prepared, and western blotting was performed as in panel A. The 15-kDa species specifically recognized by G213 is indicated with an asterisk. (C) Bone marrow cells obtained from an untreated guinea pig were lysed, and immunoprecipitation (IP) was performed with either control IgG (left lane) or G213 (right lane). Samples were resolved on an SDS-PAGE gel, followed by immunoblotting with G213. The 15-kDa species specifically recognized by G213 is indicated with an asterisk. (D) Cell lysates derived from HEK293T cell transfectants expressing either FLAG-tagged S100A9 or HA-tagged S100A8 as well as mock-treated cells were resolved on SDS-PAGE gels, and immunoblotting with Abs to FLAG, HA, and β-actin as well as G213 mAb was conducted.
    Figure Legend Snippet: Identification of the G213-reactive protein as S100A9 . (A) Tissue lysates derived from the lungs of untreated (left lane) and BCG-challenged (right lane) guinea pigs were resolved on SDS-PAGE, and western blotting was conducted with G213 (upper panel) and Ab to β-actin (lower panel). The 15-kDa species specifically recognized by G213 is indicated with an asterisk. The 30-kDa bands observed on both lanes may represent nonspecific signals. (B) Tissue lysates derived from the bone marrow, spleen, lymph nodes, and thymus of an untreated guinea pig were prepared, and western blotting was performed as in panel A. The 15-kDa species specifically recognized by G213 is indicated with an asterisk. (C) Bone marrow cells obtained from an untreated guinea pig were lysed, and immunoprecipitation (IP) was performed with either control IgG (left lane) or G213 (right lane). Samples were resolved on an SDS-PAGE gel, followed by immunoblotting with G213. The 15-kDa species specifically recognized by G213 is indicated with an asterisk. (D) Cell lysates derived from HEK293T cell transfectants expressing either FLAG-tagged S100A9 or HA-tagged S100A8 as well as mock-treated cells were resolved on SDS-PAGE gels, and immunoblotting with Abs to FLAG, HA, and β-actin as well as G213 mAb was conducted.

    Techniques Used: Derivative Assay, SDS Page, Western Blot, Immunoprecipitation, Expressing

    15) Product Images from "APIP, an ERBB3-binding partner, stimulates erbB2-3 heterodimer formation to promote tumorigenesis"

    Article Title: APIP, an ERBB3-binding partner, stimulates erbB2-3 heterodimer formation to promote tumorigenesis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.7802

    APIP is an essential activator of HRG-β1/ERBB3 in gastric cancer cells A. APIP-interacting proteins were purified from SNU-16 cells expressing 3xFLAG-tagged APIP by co-immunoprecipitation assay using FLAG M2 affinity gel. The bound proteins were resolved by SDS-PAGE and prepared for LC-MS/MS analysis. CBB, coomassie brilliant blue. B. Inhibition of HRG-β1-dependent ERBB3 phosphorylation and its downstream signals by APIP knockdown. Serum-starved SNU-16 control and APIP knockdown cells were treated with 10% FBS, 2 μM Insulin, 50 ng/ml IGF, 100 ng/ml EGF, 50 ng/ml HRG-β1 or 50 ng/ml FGF2 for 10 min and subjected to western blotting. C. Analysis of phospho-ERBB3 (Y1289) in a panel of gastric cancer cell lines by western blotting. D. Inhibition of ERBB3 activity by APIP knockdown in complete medium. Whole-cell extracts of SNU-16 control and APIP knockdown cells were analyzed with western blotting. E. APIP overexpression sensitizes SNU-620 cells to HRG-β1. SNU-620 control and APIP-overexpressing cells were stimulated with 2 ng/ml HRG-β1 and subjected to western blotting. Starv., serum starved. F. and G. ERBB3 knockdown inhibits cell growth and suppresses AKT and ERK1/2 activity. Cell growth (middle) and death rates (bottom) of SNU-16 control cells (shControl) or ERBB3 knockdown cells (shERBB3 #1 and #2) were assessed and analyzed by western blotting (top). The results represent mean ± S.D. ( n = 3). (F). Whole cell lysates were examined by western blotting (G). H. Enhanced HRG-β1 signaling in APIP transgenic MEFs. WT and APIP transgenic MEFs were treated with 10 ng/ml HRG-β1 and harvested for immunoprecipitation (IP) assay. Statistical significance is indicated as follows: *, P
    Figure Legend Snippet: APIP is an essential activator of HRG-β1/ERBB3 in gastric cancer cells A. APIP-interacting proteins were purified from SNU-16 cells expressing 3xFLAG-tagged APIP by co-immunoprecipitation assay using FLAG M2 affinity gel. The bound proteins were resolved by SDS-PAGE and prepared for LC-MS/MS analysis. CBB, coomassie brilliant blue. B. Inhibition of HRG-β1-dependent ERBB3 phosphorylation and its downstream signals by APIP knockdown. Serum-starved SNU-16 control and APIP knockdown cells were treated with 10% FBS, 2 μM Insulin, 50 ng/ml IGF, 100 ng/ml EGF, 50 ng/ml HRG-β1 or 50 ng/ml FGF2 for 10 min and subjected to western blotting. C. Analysis of phospho-ERBB3 (Y1289) in a panel of gastric cancer cell lines by western blotting. D. Inhibition of ERBB3 activity by APIP knockdown in complete medium. Whole-cell extracts of SNU-16 control and APIP knockdown cells were analyzed with western blotting. E. APIP overexpression sensitizes SNU-620 cells to HRG-β1. SNU-620 control and APIP-overexpressing cells were stimulated with 2 ng/ml HRG-β1 and subjected to western blotting. Starv., serum starved. F. and G. ERBB3 knockdown inhibits cell growth and suppresses AKT and ERK1/2 activity. Cell growth (middle) and death rates (bottom) of SNU-16 control cells (shControl) or ERBB3 knockdown cells (shERBB3 #1 and #2) were assessed and analyzed by western blotting (top). The results represent mean ± S.D. ( n = 3). (F). Whole cell lysates were examined by western blotting (G). H. Enhanced HRG-β1 signaling in APIP transgenic MEFs. WT and APIP transgenic MEFs were treated with 10 ng/ml HRG-β1 and harvested for immunoprecipitation (IP) assay. Statistical significance is indicated as follows: *, P

    Techniques Used: Purification, Expressing, Co-Immunoprecipitation Assay, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Inhibition, Western Blot, Activity Assay, Over Expression, Transgenic Assay, Immunoprecipitation

    16) Product Images from "Mapping sites of herpes simplex virus type 1 glycoprotein D that permit insertions and impact gD and gB receptors usage"

    Article Title: Mapping sites of herpes simplex virus type 1 glycoprotein D that permit insertions and impact gD and gB receptors usage

    Journal: Scientific Reports

    doi: 10.1038/srep43712

    Expression of gD mutants. ( A ) Cell surface expression measured by CELISA. CHO-K1 cells in a 96-well plate were transfected overnight with plasmids encoding FLAG-tagged gD (F-gD), gD mutants, or an empty vector. The cells were washed and incubated with an anti-FLAG M2 antibody. After extensive washing, cells were fixed and incubated with an anti-mouse secondary antibody for detection. Each bar shows the mean and standard deviation of three independent determinations. Background signals (vector control) were subtracted from the values. Data for each set of glycoproteins were normalized to the expression level of F-gD. ( B ) Total protein expression measured by western blot of cell lysates. CHO cells expressing the constructs above were lysed and proteins were resolved by SDS-PAGE. Proteins were transferred to nitrocellulose and probed with rabbit anti-FLAG antibody followed by goat anti-rabbit IgG. All mutants migrated to their predicted molecular weights.
    Figure Legend Snippet: Expression of gD mutants. ( A ) Cell surface expression measured by CELISA. CHO-K1 cells in a 96-well plate were transfected overnight with plasmids encoding FLAG-tagged gD (F-gD), gD mutants, or an empty vector. The cells were washed and incubated with an anti-FLAG M2 antibody. After extensive washing, cells were fixed and incubated with an anti-mouse secondary antibody for detection. Each bar shows the mean and standard deviation of three independent determinations. Background signals (vector control) were subtracted from the values. Data for each set of glycoproteins were normalized to the expression level of F-gD. ( B ) Total protein expression measured by western blot of cell lysates. CHO cells expressing the constructs above were lysed and proteins were resolved by SDS-PAGE. Proteins were transferred to nitrocellulose and probed with rabbit anti-FLAG antibody followed by goat anti-rabbit IgG. All mutants migrated to their predicted molecular weights.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Incubation, Standard Deviation, Western Blot, Construct, SDS Page

    17) Product Images from "The c-FLIP-NH2 terminus (p22-FLIP) induces NF-?B activation"

    Article Title: The c-FLIP-NH2 terminus (p22-FLIP) induces NF-?B activation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20051556

    p22-FLIP induces NF-κB by direct interaction with the IKK complex. (A) 293T cells were cotransfected with luciferase reporter plasmid and either MEKK1, p22-FLIP, or c-FLIP L (top part of the diagram). 293T cells were cotransfected with p22-FLIP, the luciferase reporter plasmid, and any one of the constructs IκBα, IκBβ, WT-IKKα, WT-IKKβ, mutated IKKα, or IKKβ (bottom part of the diagram). Transfection efficiency was examined using GFP transfections. NF-κB luciferase activity was determined as described in Materials and methods. (B) FLAG or FLIP immunoprecipitations were performed from 293T cells that were transfected with p22-FLIP and any one of the constructs FLAG-IKKα, FLAG-IKKβ, or FLAG-IKKγ. Immunoprecipitated products were subjected to 12% SDS-PAGE gels and analyzed by Western blot using anti-FLIP mAb NF6 and anti-FLAG mAb. (C) 293T cells were cotransfected with MEKK1, p22-FLIP, c-FLIP L , p43-FLIP, and the luciferase reporter plasmid. Transfected cells were incubated for 16 h in the presence of the indicated concentrations of zVAD-fmk and lysed, and NF-κB luciferase activity was determined.
    Figure Legend Snippet: p22-FLIP induces NF-κB by direct interaction with the IKK complex. (A) 293T cells were cotransfected with luciferase reporter plasmid and either MEKK1, p22-FLIP, or c-FLIP L (top part of the diagram). 293T cells were cotransfected with p22-FLIP, the luciferase reporter plasmid, and any one of the constructs IκBα, IκBβ, WT-IKKα, WT-IKKβ, mutated IKKα, or IKKβ (bottom part of the diagram). Transfection efficiency was examined using GFP transfections. NF-κB luciferase activity was determined as described in Materials and methods. (B) FLAG or FLIP immunoprecipitations were performed from 293T cells that were transfected with p22-FLIP and any one of the constructs FLAG-IKKα, FLAG-IKKβ, or FLAG-IKKγ. Immunoprecipitated products were subjected to 12% SDS-PAGE gels and analyzed by Western blot using anti-FLIP mAb NF6 and anti-FLAG mAb. (C) 293T cells were cotransfected with MEKK1, p22-FLIP, c-FLIP L , p43-FLIP, and the luciferase reporter plasmid. Transfected cells were incubated for 16 h in the presence of the indicated concentrations of zVAD-fmk and lysed, and NF-κB luciferase activity was determined.

    Techniques Used: Luciferase, Plasmid Preparation, Construct, Transfection, Activity Assay, Immunoprecipitation, SDS Page, Western Blot, Incubation

    p22-FLIP is generated by procaspase-8 and inhibits death receptor–induced apoptosis. (A) Procaspase-8 was immunoprecipitated from HUT78 cells using anti–caspase-8 mAb C15 and then incubated for 1 h at 37°C together with in vitro–translated [ 35 S]-labeled c-FLIP L in the presence or absence of zVAD-fmk. c-FLIP processing was analyzed by autoradiography (top left). c-FLIP cleavage products p22 and p33 are indicated. Afterward, the same membrane was subjected to Western blot analysis using anti–caspase-8 mAb C15 (bottom left). [ 35 S]-labeled c-FLIP L was incubated with the indicated concentrations of recombinant caspase-8 for 1 h at 37°C. c-FLIP processing was analyzed byautoradiography (top right). c-FLIP cleavage products p12 and p43 are indicated. Afterward, the same membrane was subjected to Western blot analysis using anti–caspase-8 mAb C15 (bottom right). (B) Analysis of p22-FLIP expression in BJAB cell lines stably overexpressing high or low amounts of p22-FLIP (p22-FLIP high or p22-FLIP low , respectively). Endogenous expression of c-FLIP S is used as a loading control. (C) p22-FLIP high , p22-FLIP low , and vector-transfected BJABs (Ctrl.) were stimulated with 1 μg/ml anti–APO-1 antibodies or 50 μl/ml LZ-CD95L for 16 h. Specific cell death was calculated as described in Materials and methods. (D) p22-FLIP high , p22-FLIP low , and vector-transfected BJABs (Ctrl.) were stimulated with the indicated concentrations of FLAG-TRAIL for 16 h. (E) CD95 DISCs were immunoprecipitated from 5 × 10 7 cells of p22-FLIP high and vector-transfected BJABs (Ctrl.) and analyzed by Western blot with anti–caspase-8 mAb C15, anti-FLIP mAb NF6, anti-CD95 polyclonal antibody C20, and anti-FADD mAb.
    Figure Legend Snippet: p22-FLIP is generated by procaspase-8 and inhibits death receptor–induced apoptosis. (A) Procaspase-8 was immunoprecipitated from HUT78 cells using anti–caspase-8 mAb C15 and then incubated for 1 h at 37°C together with in vitro–translated [ 35 S]-labeled c-FLIP L in the presence or absence of zVAD-fmk. c-FLIP processing was analyzed by autoradiography (top left). c-FLIP cleavage products p22 and p33 are indicated. Afterward, the same membrane was subjected to Western blot analysis using anti–caspase-8 mAb C15 (bottom left). [ 35 S]-labeled c-FLIP L was incubated with the indicated concentrations of recombinant caspase-8 for 1 h at 37°C. c-FLIP processing was analyzed byautoradiography (top right). c-FLIP cleavage products p12 and p43 are indicated. Afterward, the same membrane was subjected to Western blot analysis using anti–caspase-8 mAb C15 (bottom right). (B) Analysis of p22-FLIP expression in BJAB cell lines stably overexpressing high or low amounts of p22-FLIP (p22-FLIP high or p22-FLIP low , respectively). Endogenous expression of c-FLIP S is used as a loading control. (C) p22-FLIP high , p22-FLIP low , and vector-transfected BJABs (Ctrl.) were stimulated with 1 μg/ml anti–APO-1 antibodies or 50 μl/ml LZ-CD95L for 16 h. Specific cell death was calculated as described in Materials and methods. (D) p22-FLIP high , p22-FLIP low , and vector-transfected BJABs (Ctrl.) were stimulated with the indicated concentrations of FLAG-TRAIL for 16 h. (E) CD95 DISCs were immunoprecipitated from 5 × 10 7 cells of p22-FLIP high and vector-transfected BJABs (Ctrl.) and analyzed by Western blot with anti–caspase-8 mAb C15, anti-FLIP mAb NF6, anti-CD95 polyclonal antibody C20, and anti-FADD mAb.

    Techniques Used: Generated, Immunoprecipitation, Incubation, In Vitro, Labeling, Autoradiography, Western Blot, Recombinant, Expressing, Stable Transfection, Plasmid Preparation, Transfection

    18) Product Images from "GANP-mediated Recruitment of Activation-induced Cytidine Deaminase to Cell Nuclei and to Immunoglobulin Variable Region DNA *"

    Article Title: GANP-mediated Recruitment of Activation-induced Cytidine Deaminase to Cell Nuclei and to Immunoglobulin Variable Region DNA *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.131441

    GANP interacts with AID in vivo and in vitro . A , AID co-IP with GANP. Lysates from COS-7 cells, transfected with GFP-GANP, AID, GFP-GANP + AID, or GFP-alone constructs, were immunoprecipitated using anti-GFP Ab. Immunoblotting ( IB ) was carried out with anti-GFP Ab, anti-AID Ab, or anti-β-actin Ab by IP-Western blot analysis. Specific association in the IP with the anti-GFP Ab was confirmed by comparing with experiments using the control IgG ( bottom panel ). Western blots for whole cell lysates ( WCL ) are shown. B , reverse IP-Western blot analysis was performed by IP with anti-AID Abs followed by immunoblot with anti-GFP Ab or anti-AID Ab. The specific signal representing 240-kDa GFP-GANP was detected in immunoprecipitates using both mouse anti-AID Abs ( M mAb ) and rabbit anti-AID Abs ( R mAb ). The anti-AID (M) mAb contains the IgL band (25-kDa) used for IP as indicated with an asterisk. C , the WCL from COS-7 cells transfected with GFP-GANP and AID constructs was incubated for 10 min with either RNase A (5 μg/ml (+ R ) or 250 μg/ml (++ R )) or DNase I (10 units (+ D ) or 50 units (++ D )) at 4 °C or 37 °C and then immunoprecipitated with anti-GFP Ab followed by immunoblot with anti-GFP or anti-AID (M) Ab. D , interaction of endogenous GANP and AID was examined in Ramos B-cells by co-IP. The asterisk shows the IgL band. E and F , FLAG-GANP and AID proteins were synthesized in vitro using a WGE cell-free system, and coimmunoprecipitated using anti-AID (M) Ab ( E ) or anti-FLAG Ab ( F ). Controls for specificity of co-IP experiments using mouse ( M ) IgG are shown in the bottom panels ( E and F ). G , the interaction between WGE-produced GANP and AID was still preserved after treatment with RNase A or DNase I. The data were determined from three independent experiments.
    Figure Legend Snippet: GANP interacts with AID in vivo and in vitro . A , AID co-IP with GANP. Lysates from COS-7 cells, transfected with GFP-GANP, AID, GFP-GANP + AID, or GFP-alone constructs, were immunoprecipitated using anti-GFP Ab. Immunoblotting ( IB ) was carried out with anti-GFP Ab, anti-AID Ab, or anti-β-actin Ab by IP-Western blot analysis. Specific association in the IP with the anti-GFP Ab was confirmed by comparing with experiments using the control IgG ( bottom panel ). Western blots for whole cell lysates ( WCL ) are shown. B , reverse IP-Western blot analysis was performed by IP with anti-AID Abs followed by immunoblot with anti-GFP Ab or anti-AID Ab. The specific signal representing 240-kDa GFP-GANP was detected in immunoprecipitates using both mouse anti-AID Abs ( M mAb ) and rabbit anti-AID Abs ( R mAb ). The anti-AID (M) mAb contains the IgL band (25-kDa) used for IP as indicated with an asterisk. C , the WCL from COS-7 cells transfected with GFP-GANP and AID constructs was incubated for 10 min with either RNase A (5 μg/ml (+ R ) or 250 μg/ml (++ R )) or DNase I (10 units (+ D ) or 50 units (++ D )) at 4 °C or 37 °C and then immunoprecipitated with anti-GFP Ab followed by immunoblot with anti-GFP or anti-AID (M) Ab. D , interaction of endogenous GANP and AID was examined in Ramos B-cells by co-IP. The asterisk shows the IgL band. E and F , FLAG-GANP and AID proteins were synthesized in vitro using a WGE cell-free system, and coimmunoprecipitated using anti-AID (M) Ab ( E ) or anti-FLAG Ab ( F ). Controls for specificity of co-IP experiments using mouse ( M ) IgG are shown in the bottom panels ( E and F ). G , the interaction between WGE-produced GANP and AID was still preserved after treatment with RNase A or DNase I. The data were determined from three independent experiments.

    Techniques Used: In Vivo, In Vitro, Co-Immunoprecipitation Assay, Transfection, Construct, Immunoprecipitation, Western Blot, Incubation, Synthesized, Produced

    19) Product Images from "Caspase-3-mediated cleavage of p65/RelA results in a carboxy-terminal fragment that inhibits I?B? and enhances HIV-1 replication in human T lymphocytes"

    Article Title: Caspase-3-mediated cleavage of p65/RelA results in a carboxy-terminal fragment that inhibits I?B? and enhances HIV-1 replication in human T lymphocytes

    Journal: Retrovirology

    doi: 10.1186/1742-4690-5-109

    Dimerization of ΔNH 2 p65 in Jurkat cells . (a) Schematic representation of ΔNH 2 p65-tag mutant, which carries the ATG codon at Asp 97 . This mutant lacks part of DNA contact domains but not the dimerization domain. (b, c) Ten micrograms of cytosolic extracts from Jurkat cells transiently transfected with either pCMV-p65wt-tag, pCMV-p65 D94E;D97E-tag or pCMV-ΔNH 2 p65-tag expression vectors were analyzed by immunoblotting using an antibody against the carboxy-terminus of p65/RelA (b) and anti-FLAG tag M2 mAb (c). Untagged plasmid pCMV-p65wt was used as a control of the anti-FLAG tag M2 mAb specificity. (d) Two hundred micrograms of protein extracts from Jurkat cells transiently transfected with pCMV-p65wt-tag, pCMV-p65 D94E;D97E-tag and pCMV-ΔNH 2 p65-tag expression vectors were subjected to immunoprecipitation using the anti-FLAG tag M2 mAb. Analysis was carried out by immunoblotting using antibodies against the carboxy-terminus of p65/RelA, NF-κB1/p50 and IκBα. Images correspond to the same western blot gel that was first blotted simultaneously with antibodies against p65/Rel and IκBα and then it was deshybridized and reprobed with anti-NF-κB1/p50.
    Figure Legend Snippet: Dimerization of ΔNH 2 p65 in Jurkat cells . (a) Schematic representation of ΔNH 2 p65-tag mutant, which carries the ATG codon at Asp 97 . This mutant lacks part of DNA contact domains but not the dimerization domain. (b, c) Ten micrograms of cytosolic extracts from Jurkat cells transiently transfected with either pCMV-p65wt-tag, pCMV-p65 D94E;D97E-tag or pCMV-ΔNH 2 p65-tag expression vectors were analyzed by immunoblotting using an antibody against the carboxy-terminus of p65/RelA (b) and anti-FLAG tag M2 mAb (c). Untagged plasmid pCMV-p65wt was used as a control of the anti-FLAG tag M2 mAb specificity. (d) Two hundred micrograms of protein extracts from Jurkat cells transiently transfected with pCMV-p65wt-tag, pCMV-p65 D94E;D97E-tag and pCMV-ΔNH 2 p65-tag expression vectors were subjected to immunoprecipitation using the anti-FLAG tag M2 mAb. Analysis was carried out by immunoblotting using antibodies against the carboxy-terminus of p65/RelA, NF-κB1/p50 and IκBα. Images correspond to the same western blot gel that was first blotted simultaneously with antibodies against p65/Rel and IκBα and then it was deshybridized and reprobed with anti-NF-κB1/p50.

    Techniques Used: Mutagenesis, Transfection, Expressing, FLAG-tag, Plasmid Preparation, Immunoprecipitation, Western Blot

    Binding affinity assay of p65wt-tag and ΔNH 2 p65-tag to IκBα by using in vitro translated proteins . (a) One microgram of in vitro translated p65wt-tag, ΔNH 2 p65-tag and IκBα were analyzed by immunoblotting using the anti-FLAG tag M2 mAb and an antibody against IκBα. (b) The immunoprecipitation assays of p65wt-tag and ΔNH 2 p65-tag proteins – alone or combined at different rates – were carried out using a polyclonal antibody against IκBα. Immunoblotting was performed with monoclonal antibodies anti-FLAG and anti-IκBα (10B). Gel bands were quantified by densitometry and background noise was subtracted from the images. Relative ratio of optical density units was calculated regarding to the gel band with less optical density for each condition.
    Figure Legend Snippet: Binding affinity assay of p65wt-tag and ΔNH 2 p65-tag to IκBα by using in vitro translated proteins . (a) One microgram of in vitro translated p65wt-tag, ΔNH 2 p65-tag and IκBα were analyzed by immunoblotting using the anti-FLAG tag M2 mAb and an antibody against IκBα. (b) The immunoprecipitation assays of p65wt-tag and ΔNH 2 p65-tag proteins – alone or combined at different rates – were carried out using a polyclonal antibody against IκBα. Immunoblotting was performed with monoclonal antibodies anti-FLAG and anti-IκBα (10B). Gel bands were quantified by densitometry and background noise was subtracted from the images. Relative ratio of optical density units was calculated regarding to the gel band with less optical density for each condition.

    Techniques Used: Binding Assay, In Vitro, FLAG-tag, Immunoprecipitation

    Dose-effect curve of p65wt-tag and ΔNH 2 p65-tag proteins expressed separately and together by transfection in 3T3-p65ko cells . (a) 3T3-p65ko cells were transiently co-transfected with pκB-conA-LUC plasmid and both pCMV-p65wt-tag and pCMV-ΔNH 2 p65-tag expression vectors separately or combined in different ratios: pCMV-p65wt-tag expression vector was transfected at 1 μg/million of cells whereas pCMV-ΔNH 2 p65-tag expression vector was transfected at 0.5, 1 and 4 μg/million of cells (ratio p65wt/ΔNH 2 p65 2:1, 1:1, and 1:4, respectively). Luciferase expression was then analyzed in the whole protein extracts. Internal control of transfection was carried out by co-transfection with pSV-β-Galactosidase vector and protein concentration was also measured to normalize the data. The mean was performed with results from three different experiments and standard deviation is shown as a line on the top of the bars. (b) One hundred micrograms of nuclear protein extracts from 3T3-p65ko cells transiently transfected with pCMV-p65wt-tag and pCMV-ΔNH 2 p65-tag expression vectors – separately and combined in the ratio 1:4 – were subject to immunoprecipitation with the anti-FLAG tag M2 mAb. Analysis was carried out by immunoblotting with antibodies against the carboxy-terminus of p65/RelA and IκBα.
    Figure Legend Snippet: Dose-effect curve of p65wt-tag and ΔNH 2 p65-tag proteins expressed separately and together by transfection in 3T3-p65ko cells . (a) 3T3-p65ko cells were transiently co-transfected with pκB-conA-LUC plasmid and both pCMV-p65wt-tag and pCMV-ΔNH 2 p65-tag expression vectors separately or combined in different ratios: pCMV-p65wt-tag expression vector was transfected at 1 μg/million of cells whereas pCMV-ΔNH 2 p65-tag expression vector was transfected at 0.5, 1 and 4 μg/million of cells (ratio p65wt/ΔNH 2 p65 2:1, 1:1, and 1:4, respectively). Luciferase expression was then analyzed in the whole protein extracts. Internal control of transfection was carried out by co-transfection with pSV-β-Galactosidase vector and protein concentration was also measured to normalize the data. The mean was performed with results from three different experiments and standard deviation is shown as a line on the top of the bars. (b) One hundred micrograms of nuclear protein extracts from 3T3-p65ko cells transiently transfected with pCMV-p65wt-tag and pCMV-ΔNH 2 p65-tag expression vectors – separately and combined in the ratio 1:4 – were subject to immunoprecipitation with the anti-FLAG tag M2 mAb. Analysis was carried out by immunoblotting with antibodies against the carboxy-terminus of p65/RelA and IκBα.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Luciferase, Cotransfection, Protein Concentration, Standard Deviation, Immunoprecipitation, FLAG-tag

    Increase of HIV-1 replication in resting PBLs after over-expression of ΔNH 2 p65-tag . (a) Resting PBLs were transfected with pNL4.3-Renilla (a) or pNL4.3-wt (b) vectors together with pCMV-p65wt-tag and pCMV-ΔNH 2 p65-tag expression vectors, separately or in ratio 2:1, 1:1, and 1:4. Cells were maintained in culture in the absence of activation for 72 hours and then HIV-1 replication was assessed by quantification of Renilla RLUs in whole protein extracts or HIV-1 p24-gag antigen in culture supernatants. Internal control of transfection was carried out by co-transfection of the pSV-β-Galactosidase vector. Data correspond to the mean of three different experiments and lines on the top of the bars represent the standard deviation. (b) Two hundred micrograms of cytosolic and nuclear extracts from resting PBLs transfected with the control plasmid pCMV-Tag1 or pCMV-p65wt-tag and pCMV-ΔNH 2 p65-tag expression vectors were analyzed by immunoprecipitation with the anti-FLAG tag M2 mAb and subsequent immunoblotting with an antibody against the carboxy terminus of p65/RelA.
    Figure Legend Snippet: Increase of HIV-1 replication in resting PBLs after over-expression of ΔNH 2 p65-tag . (a) Resting PBLs were transfected with pNL4.3-Renilla (a) or pNL4.3-wt (b) vectors together with pCMV-p65wt-tag and pCMV-ΔNH 2 p65-tag expression vectors, separately or in ratio 2:1, 1:1, and 1:4. Cells were maintained in culture in the absence of activation for 72 hours and then HIV-1 replication was assessed by quantification of Renilla RLUs in whole protein extracts or HIV-1 p24-gag antigen in culture supernatants. Internal control of transfection was carried out by co-transfection of the pSV-β-Galactosidase vector. Data correspond to the mean of three different experiments and lines on the top of the bars represent the standard deviation. (b) Two hundred micrograms of cytosolic and nuclear extracts from resting PBLs transfected with the control plasmid pCMV-Tag1 or pCMV-p65wt-tag and pCMV-ΔNH 2 p65-tag expression vectors were analyzed by immunoprecipitation with the anti-FLAG tag M2 mAb and subsequent immunoblotting with an antibody against the carboxy terminus of p65/RelA.

    Techniques Used: Over Expression, Transfection, Expressing, Activation Assay, Cotransfection, Plasmid Preparation, Standard Deviation, Immunoprecipitation, FLAG-tag

    Subcellular localization of tagged p65/RelA and endogenous p65/RelA in activated Jurkat cells . (a) Jurkat cells did not show cleavage of p65/RelA in the cytosol or in the nucleus even after activation with PMA, as was determined by immunoblotting with an antibody against the carboxy terminus of p65/RelA. (b, c) Jurkat cells were transiently transfected with pCMV-p65wt-tag expression vector and then stimulated with PMA immediately after transfection. Analysis of protein expression was performed 18 hours after transfection by immunoblotting using an antibody against the carboxy-terminus of p65/RelA in the cytosol (b) or in the nucleus (c). Gel bands were quantified by densitometry and background noise was subtracted from the images. Relative ratio of optical density units was calculated regarding to the gel band with less optical density. (d) Analysis of subcellular distribution of tagged p65/RelA was also determined by confocal microscopy. Cells were transiently transfected with 1 μg of pCMV-p65wt-tag expression vector per million of cells and PMA or PHA was added immediately after transfection. After 18 hours, analysis of tagged protein expression was performed by confocal microscopy after staining with anti-FLAG tag M2 mAb and a secondary antibody conjugated with TexasRed. Two Jurkat cells from each experimental point related to two independent experiments are shown. (e) Analysis of the subcellular distribution of endogenous p65/RelA in Jurkat cells transiently transfected with 1 μg of pCMV-Tag1 control vector per million of cells and activated with PMA or PHA immediately after transfection. After 18 hours, analysis of p65/RelA distribution was performed by confocal microscopy after staining with an antibody against the carboxy-terminus of p65/RelA and a secondary antibody conjugated with Alexa 488. Two cells from each experimental point related to two independent experiments are shown.
    Figure Legend Snippet: Subcellular localization of tagged p65/RelA and endogenous p65/RelA in activated Jurkat cells . (a) Jurkat cells did not show cleavage of p65/RelA in the cytosol or in the nucleus even after activation with PMA, as was determined by immunoblotting with an antibody against the carboxy terminus of p65/RelA. (b, c) Jurkat cells were transiently transfected with pCMV-p65wt-tag expression vector and then stimulated with PMA immediately after transfection. Analysis of protein expression was performed 18 hours after transfection by immunoblotting using an antibody against the carboxy-terminus of p65/RelA in the cytosol (b) or in the nucleus (c). Gel bands were quantified by densitometry and background noise was subtracted from the images. Relative ratio of optical density units was calculated regarding to the gel band with less optical density. (d) Analysis of subcellular distribution of tagged p65/RelA was also determined by confocal microscopy. Cells were transiently transfected with 1 μg of pCMV-p65wt-tag expression vector per million of cells and PMA or PHA was added immediately after transfection. After 18 hours, analysis of tagged protein expression was performed by confocal microscopy after staining with anti-FLAG tag M2 mAb and a secondary antibody conjugated with TexasRed. Two Jurkat cells from each experimental point related to two independent experiments are shown. (e) Analysis of the subcellular distribution of endogenous p65/RelA in Jurkat cells transiently transfected with 1 μg of pCMV-Tag1 control vector per million of cells and activated with PMA or PHA immediately after transfection. After 18 hours, analysis of p65/RelA distribution was performed by confocal microscopy after staining with an antibody against the carboxy-terminus of p65/RelA and a secondary antibody conjugated with Alexa 488. Two cells from each experimental point related to two independent experiments are shown.

    Techniques Used: Activation Assay, Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Staining, FLAG-tag

    20) Product Images from "Identification and Characterization of CD300H, a New Member of the Human CD300 Immunoreceptor Family *"

    Article Title: Identification and Characterization of CD300H, a New Member of the Human CD300 Immunoreceptor Family *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.643361

    Biochemical analyses of CD300H. A , BW5147 cells and transfectants expressing FLAG-tagged CD300H (5 × 10 6 cells/experiment) were lysed in 1% Nonidet P-40 buffer, immunoprecipitated ( IP ) with anti-FLAG, and immunoblotted ( IB ) with anti-FLAG. B , BW5147 transfectants simultaneously expressing FLAG-CD300H and HA-CD300H were lysed in digitonin buffer, immunoprecipitated with anti-HA or anti-FLAG, and immunoblotted with anti-HA or anti-FLAG in reducing conditions. C , culture supernatant from 293T cells transiently expressing FLAG-tagged CD300Hs or mock were immunoprecipitated with anti-FLAG and immunoblotted with anti-FLAG. D , U937 cells and transfectants stably expressing FLAG-tagged CD300H were lysed in digitonin buffer, immunoprecipitated with anti-FLAG, anti-DAP12, anti-DAP10, or anti-FcϵRIγ, and immunoblotted with anti-FLAG, anti-DAP12, anti-DAP10, or anti-FcϵRIγ. Data are representative of two independent experiments.
    Figure Legend Snippet: Biochemical analyses of CD300H. A , BW5147 cells and transfectants expressing FLAG-tagged CD300H (5 × 10 6 cells/experiment) were lysed in 1% Nonidet P-40 buffer, immunoprecipitated ( IP ) with anti-FLAG, and immunoblotted ( IB ) with anti-FLAG. B , BW5147 transfectants simultaneously expressing FLAG-CD300H and HA-CD300H were lysed in digitonin buffer, immunoprecipitated with anti-HA or anti-FLAG, and immunoblotted with anti-HA or anti-FLAG in reducing conditions. C , culture supernatant from 293T cells transiently expressing FLAG-tagged CD300Hs or mock were immunoprecipitated with anti-FLAG and immunoblotted with anti-FLAG. D , U937 cells and transfectants stably expressing FLAG-tagged CD300H were lysed in digitonin buffer, immunoprecipitated with anti-FLAG, anti-DAP12, anti-DAP10, or anti-FcϵRIγ, and immunoblotted with anti-FLAG, anti-DAP12, anti-DAP10, or anti-FcϵRIγ. Data are representative of two independent experiments.

    Techniques Used: Expressing, Immunoprecipitation, Stable Transfection

    21) Product Images from "Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts"

    Article Title: Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

    Journal: Virology

    doi: 10.1016/j.virol.2011.12.009

    Expression and detection of GP4 in HeLa cells. (A) Cells were transfected with pEGFP-GP4 for 24 h (lane 4) and the total cell lysates were subjected to western-blot using anti-GFP rabbit antibody. For GFP expression, 2.0, 1.0, 0.5 μg of pEGFP-N1 (Clontech) was individually transfected for differential expression (lanes 1, 2, and 3). pcDNA3.0 (Invitrogen) which contains the identical promoter to that of pEGFP-N1 (Clontech) was used to make up the total DNA of 2.0 μg. The PRRSV N gene fused with EGFP (pEGFP-N; Rowland et al., 2003 ) served as an additional control (lane 5) and β-actin served as the loading control. (B) Western blot of the total lysates of HeLa cells transfected with pXJ41-Flag-GP4 (lane 3). HeLa cells were mock-transfected (lane 1) or transfected with the empty vector pXJ41 (lane 2) as controls. HeLa cells constitutively express human DAF readily detectable by anti-DAF MAb (Sigma-Aldrich), and this protein served as a positive control of GPI-anchored DAF (lane 4).
    Figure Legend Snippet: Expression and detection of GP4 in HeLa cells. (A) Cells were transfected with pEGFP-GP4 for 24 h (lane 4) and the total cell lysates were subjected to western-blot using anti-GFP rabbit antibody. For GFP expression, 2.0, 1.0, 0.5 μg of pEGFP-N1 (Clontech) was individually transfected for differential expression (lanes 1, 2, and 3). pcDNA3.0 (Invitrogen) which contains the identical promoter to that of pEGFP-N1 (Clontech) was used to make up the total DNA of 2.0 μg. The PRRSV N gene fused with EGFP (pEGFP-N; Rowland et al., 2003 ) served as an additional control (lane 5) and β-actin served as the loading control. (B) Western blot of the total lysates of HeLa cells transfected with pXJ41-Flag-GP4 (lane 3). HeLa cells were mock-transfected (lane 1) or transfected with the empty vector pXJ41 (lane 2) as controls. HeLa cells constitutively express human DAF readily detectable by anti-DAF MAb (Sigma-Aldrich), and this protein served as a positive control of GPI-anchored DAF (lane 4).

    Techniques Used: Expressing, Transfection, Western Blot, Plasmid Preparation, Positive Control

    Co-localization of PRRSV GP4 and the DAF (CD55) protein as a lipid raft marker on the plasma membrane of HeLa cell. Cells were transfected with pXJ41-Flag-GP4 (D through F) and at 24 h post-transfection, washed with ice-cold PBS. Cells were then co-stained with DAF-specific MAb EVR1 (anti-CD55; A and D) and rabbit anti-Flag antibody (B and E), followed by staining with Alexa Fluor 488® conjugated goat anti-mouse IgG (H + L) and Alexa Fluor 594® conjugated goat anti-rabbit IgG (H + L) secondary antibodies, respectively. Images were visualized using a laser-scanning confocal fluorescence microscope (model BX50, Olympus). Panels G through I represent the enlargement of the indicated areas in panels D through F, respectively.
    Figure Legend Snippet: Co-localization of PRRSV GP4 and the DAF (CD55) protein as a lipid raft marker on the plasma membrane of HeLa cell. Cells were transfected with pXJ41-Flag-GP4 (D through F) and at 24 h post-transfection, washed with ice-cold PBS. Cells were then co-stained with DAF-specific MAb EVR1 (anti-CD55; A and D) and rabbit anti-Flag antibody (B and E), followed by staining with Alexa Fluor 488® conjugated goat anti-mouse IgG (H + L) and Alexa Fluor 594® conjugated goat anti-rabbit IgG (H + L) secondary antibodies, respectively. Images were visualized using a laser-scanning confocal fluorescence microscope (model BX50, Olympus). Panels G through I represent the enlargement of the indicated areas in panels D through F, respectively.

    Techniques Used: Marker, Transfection, Staining, Fluorescence, Microscopy

    22) Product Images from "Dual-specificity phosphatase 23 mediates GCM1 dephosphorylation and activation"

    Article Title: Dual-specificity phosphatase 23 mediates GCM1 dephosphorylation and activation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq838

    Characterization of the interaction between DUSP23 and GCM1. ( A ) Analysis of DUSP23-GCM1 interaction by coimmunoprecipitation. 293T cells were transfected with 1 µg of pHA-GCM1 and 1 µg of pDUSP23-FLAG for 48 h and then harvested for immunoprecipitation (IP) and immunoblotting (IB) with FLAG and HA mAbs. BeWo cells were subjected to immunoprecipitation with normal IgG or DUSP23 antibody, followed by immunoblotting with GCM1 antibody. Maltose-conjugated agarose matrix pre-loaded with 0.5 µg of recombinant MBP or MBP-GCM1 protein was incubated with 0.1 µg of recombinant DUSP23-FLAG for pull-down analysis and immunoblotting with FLAG mAb. ( B ) Identification of the DUSP23-interacting domain in GCM1. 293T cells were transfected with 1 µg of pGal4-FLAG, wild-type or deletion mutant pGal4-GCM1-FLAG expression plasmid. At 48 h post-transfection, cell extracts were prepared and incubated with maltose-conjugated agarose matrix pre-loaded with 2 µg of recombinant MBP or MBP-DUSP23 protein for pull-down analysis and immunoblotting with FLAG mAb. The middle panel is immunoblotting of Gal4-FLAG and Gal4-GCM1-FLAG proteins in the input cell extracts. The lower panel shows Coomassie brilliant blue staining of MBP and MBP fusion proteins in pull-down assays.
    Figure Legend Snippet: Characterization of the interaction between DUSP23 and GCM1. ( A ) Analysis of DUSP23-GCM1 interaction by coimmunoprecipitation. 293T cells were transfected with 1 µg of pHA-GCM1 and 1 µg of pDUSP23-FLAG for 48 h and then harvested for immunoprecipitation (IP) and immunoblotting (IB) with FLAG and HA mAbs. BeWo cells were subjected to immunoprecipitation with normal IgG or DUSP23 antibody, followed by immunoblotting with GCM1 antibody. Maltose-conjugated agarose matrix pre-loaded with 0.5 µg of recombinant MBP or MBP-GCM1 protein was incubated with 0.1 µg of recombinant DUSP23-FLAG for pull-down analysis and immunoblotting with FLAG mAb. ( B ) Identification of the DUSP23-interacting domain in GCM1. 293T cells were transfected with 1 µg of pGal4-FLAG, wild-type or deletion mutant pGal4-GCM1-FLAG expression plasmid. At 48 h post-transfection, cell extracts were prepared and incubated with maltose-conjugated agarose matrix pre-loaded with 2 µg of recombinant MBP or MBP-DUSP23 protein for pull-down analysis and immunoblotting with FLAG mAb. The middle panel is immunoblotting of Gal4-FLAG and Gal4-GCM1-FLAG proteins in the input cell extracts. The lower panel shows Coomassie brilliant blue staining of MBP and MBP fusion proteins in pull-down assays.

    Techniques Used: Transfection, Immunoprecipitation, Recombinant, Incubation, Mutagenesis, Expressing, Plasmid Preparation, Staining

    Regulation of GCM1 phosphorylation and GCM1–DUSP23 interaction. ( A ) Forskolin (FSK) stimulates the interaction between DUSP23 and GCM1. BeWo cells were treated with or without 50 µM forskolin for 24 h, followed by immunoprecipitation with normal IgG or GCM1 antibody and immunoblotting with DUSP23 and GCM1 antibodies, respectively. Note that specific interaction between GCM1 and DUSP23 was detected in immunoprecipitation with GCM1 antibody, but not normal IgG. The levels of Ser269- and Ser275-phosphorylated GCM1 and DUSP23 proteins in mock- or forskolin-treated BeWo cells were analyzed by immunoblotting with p-Ser269275-GCM1 and DUSP23 antibodies, respectively. Arrow and asterisk indicate the positions of the Ser269- and Ser275-phosphorylated GCM1 and a non-specific protein, respectively. In a separate experiment, cells were harvested for quantitative real-time PCR analysis of DUSP23 transcript. Mean values and the SD obtained from three independent experiments are presented. ( B ) Phosphorylation of Ser269 and Ser275 by PKA enhances DUSP23-GCM1 interaction. 0.5 µg of matrix-bound MBP or MBP-GCM1(167–349) was incubated with PKA for in vitro kinase reaction and then with 0.1 µg of recombinant DUSP23-FLAG in pull-down experiments. Phosphorylation of Ser269 and Ser275 in MBP-GCM1(167–349) was analyzed by immunoblotting with p-Ser269275-GCM1 antibody (middle panel). The lower panel is Coomassie brilliant blue staining of MBP (arrowhead) and MBP-GCM1(167–349) (arrow) in pull-down assays. ( C ) Activation of cAMP/PKA signaling stimulates Ser269 and Ser275 phosphorylation and enhances the interaction between GCM1 and DUSP23 in BeWo and primary cytotrophoblast cells. BeWo or CTB cells were treated with the indicated combinations of forskolin and H89. After 24 h incubation, cells were harvested for immunoblotting with p-Ser269275-GCM1 antibody. Arrow and asterisk indicate the positions of the Ser269- and Ser275-phosphorylated GCM1 and a non-specific protein, respectively. In a separate experiment, the cell lysates were subjected to immunoprecipitation with GCM1 antibody and immunoblotting with DUSP23 antibody. Arrowhead indicates the position of DUSP23. ( D ) Mutagenesis of Ser269 and Ser275 abolishes the enhancement effect of PKA and forskolin on DUSP23-GCM1 interaction. 293T cells were transfected with the indicated expression plasmids for 24 h, followed by treatment with or without 50 µM forskolin for additional 24 h. Cells were harvested for immunoprecipitation and immunoblotting with HA and FLAG mAbs. Note that coexpression of PKA or treatment with forskolin enhances the interaction between DUSP23 and the wild-type GCM1, but not the GCM1SSAA mutant. ( E ) Enhanced interaction between DUSP23 and phospho-mimetic GCM1 mutant, GCM1SSEE. 293T cells were transfected with the indicated expression plasmids for 24 h. Cells were harvested for immunoprecipitation and immunoblotting with HA and FLAG mAbs.
    Figure Legend Snippet: Regulation of GCM1 phosphorylation and GCM1–DUSP23 interaction. ( A ) Forskolin (FSK) stimulates the interaction between DUSP23 and GCM1. BeWo cells were treated with or without 50 µM forskolin for 24 h, followed by immunoprecipitation with normal IgG or GCM1 antibody and immunoblotting with DUSP23 and GCM1 antibodies, respectively. Note that specific interaction between GCM1 and DUSP23 was detected in immunoprecipitation with GCM1 antibody, but not normal IgG. The levels of Ser269- and Ser275-phosphorylated GCM1 and DUSP23 proteins in mock- or forskolin-treated BeWo cells were analyzed by immunoblotting with p-Ser269275-GCM1 and DUSP23 antibodies, respectively. Arrow and asterisk indicate the positions of the Ser269- and Ser275-phosphorylated GCM1 and a non-specific protein, respectively. In a separate experiment, cells were harvested for quantitative real-time PCR analysis of DUSP23 transcript. Mean values and the SD obtained from three independent experiments are presented. ( B ) Phosphorylation of Ser269 and Ser275 by PKA enhances DUSP23-GCM1 interaction. 0.5 µg of matrix-bound MBP or MBP-GCM1(167–349) was incubated with PKA for in vitro kinase reaction and then with 0.1 µg of recombinant DUSP23-FLAG in pull-down experiments. Phosphorylation of Ser269 and Ser275 in MBP-GCM1(167–349) was analyzed by immunoblotting with p-Ser269275-GCM1 antibody (middle panel). The lower panel is Coomassie brilliant blue staining of MBP (arrowhead) and MBP-GCM1(167–349) (arrow) in pull-down assays. ( C ) Activation of cAMP/PKA signaling stimulates Ser269 and Ser275 phosphorylation and enhances the interaction between GCM1 and DUSP23 in BeWo and primary cytotrophoblast cells. BeWo or CTB cells were treated with the indicated combinations of forskolin and H89. After 24 h incubation, cells were harvested for immunoblotting with p-Ser269275-GCM1 antibody. Arrow and asterisk indicate the positions of the Ser269- and Ser275-phosphorylated GCM1 and a non-specific protein, respectively. In a separate experiment, the cell lysates were subjected to immunoprecipitation with GCM1 antibody and immunoblotting with DUSP23 antibody. Arrowhead indicates the position of DUSP23. ( D ) Mutagenesis of Ser269 and Ser275 abolishes the enhancement effect of PKA and forskolin on DUSP23-GCM1 interaction. 293T cells were transfected with the indicated expression plasmids for 24 h, followed by treatment with or without 50 µM forskolin for additional 24 h. Cells were harvested for immunoprecipitation and immunoblotting with HA and FLAG mAbs. Note that coexpression of PKA or treatment with forskolin enhances the interaction between DUSP23 and the wild-type GCM1, but not the GCM1SSAA mutant. ( E ) Enhanced interaction between DUSP23 and phospho-mimetic GCM1 mutant, GCM1SSEE. 293T cells were transfected with the indicated expression plasmids for 24 h. Cells were harvested for immunoprecipitation and immunoblotting with HA and FLAG mAbs.

    Techniques Used: Immunoprecipitation, Real-time Polymerase Chain Reaction, Incubation, In Vitro, Recombinant, Staining, Activation Assay, CtB Assay, Mutagenesis, Transfection, Expressing

    Regulation of GCM1 ubiquitination and half-life by DUSP23. ( A ) DUSP23 prevents GCM1 from ubiquitination. 293T cells were transfected with different combinations of 1 µg of pGCM1-FLAG, pHA-Ub, pDUSP23-Myc and pDUSP23DACS-Myc. At 24 h post-transfection, cells were treated with 40 µM MG132 for an additional 10 h, and then subjected to ubiquitination analysis by immunoprecipitation with FLAG mAb and immunoblotting with HA mAb. ( B ) DUSP23 prolongs the half-life of GCM1. Mock BeWo31 cells or BeWo31 cells stably expressing DUSP23-Myc or DUSP23DACS-Myc were treated with 75 µM cycloheximide (CHX) for the indicated period of time. The protein levels of HA-GCM1, DUSP23-Myc, DUSP23DACS-Myc and β-actin were then analyzed by immunoblotting with HA, Myc and β-actin mAbs. The band intensity of HA-GCM1 and β-actin was quantified by densitometric analysis. After normalization of HA-GCM1 with β-actin, the relative levels of HA-GCM1 proteins from two independent experiments were plotted against the time course of CHX treatment.
    Figure Legend Snippet: Regulation of GCM1 ubiquitination and half-life by DUSP23. ( A ) DUSP23 prevents GCM1 from ubiquitination. 293T cells were transfected with different combinations of 1 µg of pGCM1-FLAG, pHA-Ub, pDUSP23-Myc and pDUSP23DACS-Myc. At 24 h post-transfection, cells were treated with 40 µM MG132 for an additional 10 h, and then subjected to ubiquitination analysis by immunoprecipitation with FLAG mAb and immunoblotting with HA mAb. ( B ) DUSP23 prolongs the half-life of GCM1. Mock BeWo31 cells or BeWo31 cells stably expressing DUSP23-Myc or DUSP23DACS-Myc were treated with 75 µM cycloheximide (CHX) for the indicated period of time. The protein levels of HA-GCM1, DUSP23-Myc, DUSP23DACS-Myc and β-actin were then analyzed by immunoblotting with HA, Myc and β-actin mAbs. The band intensity of HA-GCM1 and β-actin was quantified by densitometric analysis. After normalization of HA-GCM1 with β-actin, the relative levels of HA-GCM1 proteins from two independent experiments were plotted against the time course of CHX treatment.

    Techniques Used: Transfection, Immunoprecipitation, Stable Transfection, Expressing

    Regulation of GCM1 stability by DUSP23. ( A ) The steady state level of GCM1 increased in the presence of DUSP23. 293T cells were transfected with 1 µg of pGCM1-FLAG and the indicated amount of pDUSP23-Myc or pDUSP23DACS-Myc for 48 h. Cells were harvested for immunoblotted with FLAG, Myc or β-actin mAb. The result of a short exposure time (1 min) for the level of GCM1-FLAG in 293T coexpressing DUSP23-Myc is presented in the top of the left panel. Note that the result of a longer exposure time (5 min) for the level of GCM1-FLAG in 293T cell coexpressing DUSP23DACS-Myc is presented in the top of the right panel. For comparison, the result of a short exposure time (1 min) is provided underneath. ( B ) GCM1-mediated transcriptional activation is enhanced by DUSP23. 293T cells were transfected with 0.1 µg of p(GBS) 4 E1BLuc and different combinations of 0.05 µg of pGCM1-FLAG and increasing amounts of pDUSP23-Myc or pDUSP23DACS-Myc for 48 h. Cells were then harvested for luciferase reporter assay. Mean values and the SD obtained from three independent experiments are presented. ( C ) The transcript levels of GCM1-FLAG in 293T cells coexpressing with different amounts of DUSP23-Myc were analyzed by quantitative real-time PCR. Mean values and the SD obtained from three independent experiments are presented.
    Figure Legend Snippet: Regulation of GCM1 stability by DUSP23. ( A ) The steady state level of GCM1 increased in the presence of DUSP23. 293T cells were transfected with 1 µg of pGCM1-FLAG and the indicated amount of pDUSP23-Myc or pDUSP23DACS-Myc for 48 h. Cells were harvested for immunoblotted with FLAG, Myc or β-actin mAb. The result of a short exposure time (1 min) for the level of GCM1-FLAG in 293T coexpressing DUSP23-Myc is presented in the top of the left panel. Note that the result of a longer exposure time (5 min) for the level of GCM1-FLAG in 293T cell coexpressing DUSP23DACS-Myc is presented in the top of the right panel. For comparison, the result of a short exposure time (1 min) is provided underneath. ( B ) GCM1-mediated transcriptional activation is enhanced by DUSP23. 293T cells were transfected with 0.1 µg of p(GBS) 4 E1BLuc and different combinations of 0.05 µg of pGCM1-FLAG and increasing amounts of pDUSP23-Myc or pDUSP23DACS-Myc for 48 h. Cells were then harvested for luciferase reporter assay. Mean values and the SD obtained from three independent experiments are presented. ( C ) The transcript levels of GCM1-FLAG in 293T cells coexpressing with different amounts of DUSP23-Myc were analyzed by quantitative real-time PCR. Mean values and the SD obtained from three independent experiments are presented.

    Techniques Used: Transfection, Activation Assay, Luciferase, Reporter Assay, Real-time Polymerase Chain Reaction

    Coordination of GCM1 dephosphorylation and acetylation. ( A ) Interplay between DUSP23 and CBP in regulation of GCM1 ubiquitination. 293T cells were transfected with different combinations of 2 µg of pGCM1-Myc, 0.5 µg of pHA-Ub, 1 µg of pCBP-FLAG, 2 µg of pDUSP23-Myc and 2 µg of pDUSP23DACS-Myc. At 48 h post-transfection, cells were treated with 40 µM MG132 for an additional 6 h and then subjected to immunoprecipitation and immunoblotting with GCM1 antibody, and HA, FLAG and Myc mAbs. ( B ) DUSP23-mediated GCM1 dephosphorylation facilitates CBP-mediated GCM1 acetylation. 293T cells expressing scramble or DUSP23 shRNA were transfected with 2 µg of pGCM1-Myc and 1 µg of pCBP-FLAG. At 48 h post-transfection, cells were treated with 40 µM MG132 for an additional 10 h and then subjected to immunoprecipitation and immunoblotting with GCM1 antibody, and Ac-K and Myc mAbs. Arrow indicates the position of acetylated GCM1. ( C ) Dephosphorylation of GCM1 by DUSP23 facilitates GCM1 acetylation in placental cells. BeWo31 cells expressing scramble or DUSP23 shRNA were mock-treated or treated with 10 µM forskolin and 50 ng/ml TSA for 24 h for immunoprecipitation and immunoblotting with Ac-K and HA mAbs. In a separate experiment, cells were further treated with 20 µM MG132 for additional 10 h before harvesting for the above-described analysis. The numbers underneath lanes 6 and 12 indicate the ratios of the acetylated and Ser322-phosphorylated HA-GCM1 band intensity (normalized against Ser322-phosphorylated HA-GCM1) in FSK- and TSA-treated cells to that in mock-treated cells.
    Figure Legend Snippet: Coordination of GCM1 dephosphorylation and acetylation. ( A ) Interplay between DUSP23 and CBP in regulation of GCM1 ubiquitination. 293T cells were transfected with different combinations of 2 µg of pGCM1-Myc, 0.5 µg of pHA-Ub, 1 µg of pCBP-FLAG, 2 µg of pDUSP23-Myc and 2 µg of pDUSP23DACS-Myc. At 48 h post-transfection, cells were treated with 40 µM MG132 for an additional 6 h and then subjected to immunoprecipitation and immunoblotting with GCM1 antibody, and HA, FLAG and Myc mAbs. ( B ) DUSP23-mediated GCM1 dephosphorylation facilitates CBP-mediated GCM1 acetylation. 293T cells expressing scramble or DUSP23 shRNA were transfected with 2 µg of pGCM1-Myc and 1 µg of pCBP-FLAG. At 48 h post-transfection, cells were treated with 40 µM MG132 for an additional 10 h and then subjected to immunoprecipitation and immunoblotting with GCM1 antibody, and Ac-K and Myc mAbs. Arrow indicates the position of acetylated GCM1. ( C ) Dephosphorylation of GCM1 by DUSP23 facilitates GCM1 acetylation in placental cells. BeWo31 cells expressing scramble or DUSP23 shRNA were mock-treated or treated with 10 µM forskolin and 50 ng/ml TSA for 24 h for immunoprecipitation and immunoblotting with Ac-K and HA mAbs. In a separate experiment, cells were further treated with 20 µM MG132 for additional 10 h before harvesting for the above-described analysis. The numbers underneath lanes 6 and 12 indicate the ratios of the acetylated and Ser322-phosphorylated HA-GCM1 band intensity (normalized against Ser322-phosphorylated HA-GCM1) in FSK- and TSA-treated cells to that in mock-treated cells.

    Techniques Used: De-Phosphorylation Assay, Transfection, Immunoprecipitation, Expressing, shRNA

    23) Product Images from "A mutant heterodimeric myosin with one inactive head generates maximal displacement"

    Article Title: A mutant heterodimeric myosin with one inactive head generates maximal displacement

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200304023

    Western blots of E470A/wt heterodimer. Left side reacted with anti-Flag antibody M2 (Sigma-Aldrich) and right side reacted with anti-(His) 6 -tag mAb (Sigma-Aldrich). Lanes 1–3 and 1′-3′ are purified FLAG- and (His) 6 -labeled HMM standards in loads of 25, 35, and 45 ng, respectively. Lane 4 and lane 5 on both blots are identical samples of the heterodimer containing 35 and 45 ng protein. The amount of Flag- and (His) 6 -reactive material in the heterodimer was determined by normalization to the staining intensity of the standards.
    Figure Legend Snippet: Western blots of E470A/wt heterodimer. Left side reacted with anti-Flag antibody M2 (Sigma-Aldrich) and right side reacted with anti-(His) 6 -tag mAb (Sigma-Aldrich). Lanes 1–3 and 1′-3′ are purified FLAG- and (His) 6 -labeled HMM standards in loads of 25, 35, and 45 ng, respectively. Lane 4 and lane 5 on both blots are identical samples of the heterodimer containing 35 and 45 ng protein. The amount of Flag- and (His) 6 -reactive material in the heterodimer was determined by normalization to the staining intensity of the standards.

    Techniques Used: Western Blot, Purification, Labeling, Staining

    24) Product Images from "An In Vitro Model for Lewy Body-Like Hyaline Inclusion/Astrocytic Hyaline Inclusion: Induction by ER Stress with an ALS-Linked SOD1 Mutation"

    Article Title: An In Vitro Model for Lewy Body-Like Hyaline Inclusion/Astrocytic Hyaline Inclusion: Induction by ER Stress with an ALS-Linked SOD1 Mutation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001030

    Ubiquitination of mutant SOD1 aggregates. (A–R) Colocalization assay with SOD1 and ubiquitin. SK-N-SH cells expressing wild-type SOD1 (A–I) or L84V SOD1 (J–R) were incubated with 1 µg/ml of tunicamycin (D–F, M–O), 4 µg/ml of ALLN (G–I, P–R), or no agents (A–C, J–L) for 24 h. Then the cells were fixed and stained with anti-SOD1 antibody (green; A, D, G, J, M, P) or anti-ubiquitin antibody (red; B, E, H, K, N, Q). Arrows indicate colocalization of SOD1 aggregates and ubiquitin. Scale bar = 20 µm. (S) Co-immunoprecipitation assay utilizing ubiquitin. SK-N-SH cells stably expressing wild-type and L84V SOD1 were transfected with a myc-tagged ubiquitin expression vector. After incubation with or without ALLN, cell lysates were prepared and assayed with anti-myc antibody of the immunoprecipitant with anti-FLAG antibody. Asterisk shows an ubiquitinated ladder that appeared after ALLN treatment of L84V SOD1-expressing cells. IgG bands are shown as loading controls.
    Figure Legend Snippet: Ubiquitination of mutant SOD1 aggregates. (A–R) Colocalization assay with SOD1 and ubiquitin. SK-N-SH cells expressing wild-type SOD1 (A–I) or L84V SOD1 (J–R) were incubated with 1 µg/ml of tunicamycin (D–F, M–O), 4 µg/ml of ALLN (G–I, P–R), or no agents (A–C, J–L) for 24 h. Then the cells were fixed and stained with anti-SOD1 antibody (green; A, D, G, J, M, P) or anti-ubiquitin antibody (red; B, E, H, K, N, Q). Arrows indicate colocalization of SOD1 aggregates and ubiquitin. Scale bar = 20 µm. (S) Co-immunoprecipitation assay utilizing ubiquitin. SK-N-SH cells stably expressing wild-type and L84V SOD1 were transfected with a myc-tagged ubiquitin expression vector. After incubation with or without ALLN, cell lysates were prepared and assayed with anti-myc antibody of the immunoprecipitant with anti-FLAG antibody. Asterisk shows an ubiquitinated ladder that appeared after ALLN treatment of L84V SOD1-expressing cells. IgG bands are shown as loading controls.

    Techniques Used: Mutagenesis, Expressing, Incubation, Staining, Co-Immunoprecipitation Assay, Stable Transfection, Transfection, Plasmid Preparation

    25) Product Images from "Desmuslin, an intermediate filament protein that interacts with ?-dystrobrevin and desmin"

    Article Title: Desmuslin, an intermediate filament protein that interacts with ?-dystrobrevin and desmin

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

    doi: 10.1073/pnas.111153298

    Portions of the DMN rod domain specifically interact with dystrobrevin. ( A ) CoIP experiments using the anti-FLAG antibody show that FLAG 1A-1B-2A-2B DMN specifically interacts with dystrobrevin exons 1–16 (lane 2) and 8–16 (long) (lane 3). Other regions of α-dystrobrevin do not coimmunoprecipitate in the presence of DMN (lanes 1, 4, and 5). ( B ) By varying the length of the DMN rod domain, CoIP experiments show that dystrobrevin exons 1–16 interacts with 1A-1B-2A-2B (lane 1) and 1A-1B-2A (lane 2), but not with the shorter versions of the DMN rod domain (lanes 3–5). Proteins are identified by an *.
    Figure Legend Snippet: Portions of the DMN rod domain specifically interact with dystrobrevin. ( A ) CoIP experiments using the anti-FLAG antibody show that FLAG 1A-1B-2A-2B DMN specifically interacts with dystrobrevin exons 1–16 (lane 2) and 8–16 (long) (lane 3). Other regions of α-dystrobrevin do not coimmunoprecipitate in the presence of DMN (lanes 1, 4, and 5). ( B ) By varying the length of the DMN rod domain, CoIP experiments show that dystrobrevin exons 1–16 interacts with 1A-1B-2A-2B (lane 1) and 1A-1B-2A (lane 2), but not with the shorter versions of the DMN rod domain (lanes 3–5). Proteins are identified by an *.

    Techniques Used: Co-Immunoprecipitation Assay

    CoIP analysis of DMN clones with dystrobrevin. ( A ) and DMN clones 4C, 5D, 5D-1, and 5D-2. IF designates a region with an intermediate filament signature. ( B ) When simultaneously expressed, FLAG 5D-1 coimmunoprecipitates dystrobrevin (compare lane 5 to lane 4). FLAG 5D-2 and FLAG 4C do not coimmunoprecipitate dystrobrevin (lanes 6 and 7). Our controls show that the FLAG antibody specifically precipitates proteins with the FLAG epitope (lanes 1–3) and precipitates background levels of dystrobrevin (lane 4). * indicates 35 S-labeled dystrobrevin. +/− indicates the presence or absence of a particular construct.
    Figure Legend Snippet: CoIP analysis of DMN clones with dystrobrevin. ( A ) and DMN clones 4C, 5D, 5D-1, and 5D-2. IF designates a region with an intermediate filament signature. ( B ) When simultaneously expressed, FLAG 5D-1 coimmunoprecipitates dystrobrevin (compare lane 5 to lane 4). FLAG 5D-2 and FLAG 4C do not coimmunoprecipitate dystrobrevin (lanes 6 and 7). Our controls show that the FLAG antibody specifically precipitates proteins with the FLAG epitope (lanes 1–3) and precipitates background levels of dystrobrevin (lane 4). * indicates 35 S-labeled dystrobrevin. +/− indicates the presence or absence of a particular construct.

    Techniques Used: Co-Immunoprecipitation Assay, Clone Assay, FLAG-tag, Labeling, Construct

    26) Product Images from "Ebola Virus Glycoprotein Promotes Enhanced Viral Egress by Preventing Ebola VP40 From Associating With the Host Restriction Factor BST2/Tetherin"

    Article Title: Ebola Virus Glycoprotein Promotes Enhanced Viral Egress by Preventing Ebola VP40 From Associating With the Host Restriction Factor BST2/Tetherin

    Journal: The Journal of Infectious Diseases

    doi: 10.1093/infdis/jiv125

    Although a glycoprotein (GP) molecule deleted for its mucin domain can still enhance virus-like particle (VLP) egress, it can no longer down-regulate cell surface BST2. 293T::vector or 293T::BST2-HA cells were cotransfected with both an enhanced green fluorescent protein (EGFP)–expressing construct and a construct expressing the indicated GP molecule. The cells were then stained for BST2, and flow cytometry was performed. A , Histograms represent the surface BST2 levels of EGFP-gated cells. B , VP40 VLP release. 293T::vector or 293T::BST2-HA cells were transfected with a construct expressing FLAG-VP40 along with a construct expressing the indicated GP or Vphu molecule. To quantitate VP40 levels, both total cell lysates and purified VLPs were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and FLAG immunoblots (IBs) were performed. Abbreviations: HA, hemagglutinin; sGP, secreted GP; WT, wild-type.
    Figure Legend Snippet: Although a glycoprotein (GP) molecule deleted for its mucin domain can still enhance virus-like particle (VLP) egress, it can no longer down-regulate cell surface BST2. 293T::vector or 293T::BST2-HA cells were cotransfected with both an enhanced green fluorescent protein (EGFP)–expressing construct and a construct expressing the indicated GP molecule. The cells were then stained for BST2, and flow cytometry was performed. A , Histograms represent the surface BST2 levels of EGFP-gated cells. B , VP40 VLP release. 293T::vector or 293T::BST2-HA cells were transfected with a construct expressing FLAG-VP40 along with a construct expressing the indicated GP or Vphu molecule. To quantitate VP40 levels, both total cell lysates and purified VLPs were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and FLAG immunoblots (IBs) were performed. Abbreviations: HA, hemagglutinin; sGP, secreted GP; WT, wild-type.

    Techniques Used: Plasmid Preparation, Expressing, Construct, Staining, Flow Cytometry, Cytometry, Transfection, Purification, Polyacrylamide Gel Electrophoresis, Western Blot

    BST2 levels are increased in virus-like particles (VLPs) when glycoprotein (GP) is present. A , BST2 incorporation into VP40 VLPs. The 293T::vector and 293T::BST2-HA cells were cotransfected with FLAG-VP40 and either GP, Vphu, or control expression constructs. An enhanced green fluorescent protein (EGFP)–expression construct was included as a transfection control. Cell lysates and purified VLPs were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblots (IB) for the indicated proteins were performed. B , BST2 incorporation into human immunodeficiency virus (HIV) VLPs. The 293T::vector and 293T::BST2-HA cells were transfected with the HIV Gag expression constructs and the indicated GP, Vphu, and control constructs. Cell lysates and purified VLPs were run on SDS-PAGE, and IB for the indicated proteins were performed. Abbreviations: HA, hemagglutinin; HIV, human immunodeficiency virus.
    Figure Legend Snippet: BST2 levels are increased in virus-like particles (VLPs) when glycoprotein (GP) is present. A , BST2 incorporation into VP40 VLPs. The 293T::vector and 293T::BST2-HA cells were cotransfected with FLAG-VP40 and either GP, Vphu, or control expression constructs. An enhanced green fluorescent protein (EGFP)–expression construct was included as a transfection control. Cell lysates and purified VLPs were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblots (IB) for the indicated proteins were performed. B , BST2 incorporation into human immunodeficiency virus (HIV) VLPs. The 293T::vector and 293T::BST2-HA cells were transfected with the HIV Gag expression constructs and the indicated GP, Vphu, and control constructs. Cell lysates and purified VLPs were run on SDS-PAGE, and IB for the indicated proteins were performed. Abbreviations: HA, hemagglutinin; HIV, human immunodeficiency virus.

    Techniques Used: Plasmid Preparation, Expressing, Construct, Transfection, Purification, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot

    Glycoprotein (GP) does not remove BST2 from lipid rafts or virus-like particles (VLPs). A , Lipid raft fractionation of BST2. 293T::BST2-HA cells expressing VP40 and/or GP were lysed in 0.1% Triton X-100 and then subjected to iodixanol (0%–40%) density gradient centrifugation. Fractions were collected from these gradients, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted for BST2 (IB = BST2). The top blot was stripped and reprobed for the lipid raft marker flotillin (IB = flotillin). B , BST2 incorporation into VP40 VLPs. The 293T::BST2-HA or vector cells were transfected with a construct expressing FLAG-VP40 and/or a construct expressing GP-Myc. Cell lysates and purified VLPs were run on SDS-PAGE, and immunoblots for the indicated proteins were performed. Abbreviations: EGFP, enhanced green fluorescent protein; HA, hemagglutinin.
    Figure Legend Snippet: Glycoprotein (GP) does not remove BST2 from lipid rafts or virus-like particles (VLPs). A , Lipid raft fractionation of BST2. 293T::BST2-HA cells expressing VP40 and/or GP were lysed in 0.1% Triton X-100 and then subjected to iodixanol (0%–40%) density gradient centrifugation. Fractions were collected from these gradients, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotted for BST2 (IB = BST2). The top blot was stripped and reprobed for the lipid raft marker flotillin (IB = flotillin). B , BST2 incorporation into VP40 VLPs. The 293T::BST2-HA or vector cells were transfected with a construct expressing FLAG-VP40 and/or a construct expressing GP-Myc. Cell lysates and purified VLPs were run on SDS-PAGE, and immunoblots for the indicated proteins were performed. Abbreviations: EGFP, enhanced green fluorescent protein; HA, hemagglutinin.

    Techniques Used: Fractionation, Expressing, Gradient Centrifugation, Polyacrylamide Gel Electrophoresis, SDS Page, Marker, Plasmid Preparation, Transfection, Construct, Purification, Western Blot

    27) Product Images from "Nuclear Factor NF45 Interacts with Viral Proteins of Infectious Bursal Disease Virus and Inhibits Viral Replication ▿"

    Article Title: Nuclear Factor NF45 Interacts with Viral Proteins of Infectious Bursal Disease Virus and Inhibits Viral Replication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02506-09

    Coimmunoprecipitation of NF45 and IBDV proteins after RNase treatment. Lysates of pcDNA3-ckNF45-FLAG-transfected and subsequent IBDV-infected DF-1 cells (A) or DF-1 cells only IBDV infected (B) were subjected to RNA digestion with either RNase T 1 or RNase III or double-treated (T1+ R III) for 1 h at 37°C. Untreated cells were used as controls. One portion of each sample was subsequently analyzed for RNA content on a 1% agarose gel (left panel). The remaining portions of the samples were used for immunoprecipitations (IP). IP were performed with either the anti-FLAG MAb (A) or the VP2-specific MAb R63 and the VP3-specific MAb IBDV2 (B). The resulting precipitates were separated by SDS-PAGE and analyzed by Western blotting (WB) using either rabbit anti-ckNF45-His serum (anti-VP3 or -VP1) or IBDV protein-specific antiserum (rabbit anti-IBDV serum).
    Figure Legend Snippet: Coimmunoprecipitation of NF45 and IBDV proteins after RNase treatment. Lysates of pcDNA3-ckNF45-FLAG-transfected and subsequent IBDV-infected DF-1 cells (A) or DF-1 cells only IBDV infected (B) were subjected to RNA digestion with either RNase T 1 or RNase III or double-treated (T1+ R III) for 1 h at 37°C. Untreated cells were used as controls. One portion of each sample was subsequently analyzed for RNA content on a 1% agarose gel (left panel). The remaining portions of the samples were used for immunoprecipitations (IP). IP were performed with either the anti-FLAG MAb (A) or the VP2-specific MAb R63 and the VP3-specific MAb IBDV2 (B). The resulting precipitates were separated by SDS-PAGE and analyzed by Western blotting (WB) using either rabbit anti-ckNF45-His serum (anti-VP3 or -VP1) or IBDV protein-specific antiserum (rabbit anti-IBDV serum).

    Techniques Used: Transfection, Infection, Agarose Gel Electrophoresis, SDS Page, Western Blot

    ]) or the newly established chicken anti-VP1 serum (anti-VP1 ch) and a rabbit anti-VP3 serum (anti-VP3 R). The locations of the IBDV proteins (VP) are indicated by arrows. (B) For monitoring the purification of ckNF45-His, samples were taken at different stages (L, lysate; S, supernatant after centrifugation; E, eluate) and subjected to SDS-PAGE. The gel was stained with Imperial protein stain. The eluate was monitored by using an HRP-conjugated anti-His monoclonal antibody and the rabbit ckNF45-His serum. The reactivity of the rabbit anti-ckNF45His serum was tested by Western blotting of cellular lysates from different species, namely, chicken (DF-1 and CEC), quail (QM-7), dog (MDCK), monkey (Vero), human (293T), and hamster (BHK 21). The molecular masses (in kDa) of protein markers (M) are indicated on the left. (C) BHK21 cells were transfected with a mammalian vector expressing ckNF45-FLAG (see Materials and Methods). Fixed cells were incubated with rabbit anti-ckNF45-His antiserum (NF45) and a mouse anti-FLAG MAb (FLAG) followed by incubation with appropriate conjugates (goat anti-mouse-Cy3 or goat anti-rabbit-FITC). The cellular nuclei were visualized by PI staining. An overlay of the confocal pictures is shown (merge).
    Figure Legend Snippet: ]) or the newly established chicken anti-VP1 serum (anti-VP1 ch) and a rabbit anti-VP3 serum (anti-VP3 R). The locations of the IBDV proteins (VP) are indicated by arrows. (B) For monitoring the purification of ckNF45-His, samples were taken at different stages (L, lysate; S, supernatant after centrifugation; E, eluate) and subjected to SDS-PAGE. The gel was stained with Imperial protein stain. The eluate was monitored by using an HRP-conjugated anti-His monoclonal antibody and the rabbit ckNF45-His serum. The reactivity of the rabbit anti-ckNF45His serum was tested by Western blotting of cellular lysates from different species, namely, chicken (DF-1 and CEC), quail (QM-7), dog (MDCK), monkey (Vero), human (293T), and hamster (BHK 21). The molecular masses (in kDa) of protein markers (M) are indicated on the left. (C) BHK21 cells were transfected with a mammalian vector expressing ckNF45-FLAG (see Materials and Methods). Fixed cells were incubated with rabbit anti-ckNF45-His antiserum (NF45) and a mouse anti-FLAG MAb (FLAG) followed by incubation with appropriate conjugates (goat anti-mouse-Cy3 or goat anti-rabbit-FITC). The cellular nuclei were visualized by PI staining. An overlay of the confocal pictures is shown (merge).

    Techniques Used: Purification, Centrifugation, SDS Page, Staining, Western Blot, Capillary Electrochromatography, Transfection, Plasmid Preparation, Expressing, Incubation

    NF45 interacts with viral proteins VP1, VP2, and VP3. (A) Lysates of the cytoplasmic fractions of nontransfected DF-1 cells (DF1), DF-1 cells transfected with pcDNA3-ckNF45FLAG (DF1/NF45), nontransfected but IBDV-infected DF-1 cells (DF1/D78), and pcDNA3-ckNF45-FLAG-transfected and IBDV-infected Df1 cells (Df1/NF45/D78) were analyzed either directly in a Western blot assay (WB) or were immunoprecipitated (IP) using the anti-FLAG MAb followed by Western blot analysis of the IP products. (B and C) Lysates of the cytoplasmic fractions of noninfected DF-1 cells (DF1) and IBDV-infected DF-1 cells (DF1/D78) were either directly used for WB or for IP using the VP2-specific MAb R63 (B) or the VP3-specific MAb IBDV2 (C) followed by Western blot analysis. Western blots shown in all three panels were performed using rabbit anti-ckNF45-His serum or rabbit anti-VP1, -VP2, or -VP3 serum. The detected proteins are named and marked by arrows. The molecular masses (in kilodaltons) of protein markers are indicated to the left of each gel.
    Figure Legend Snippet: NF45 interacts with viral proteins VP1, VP2, and VP3. (A) Lysates of the cytoplasmic fractions of nontransfected DF-1 cells (DF1), DF-1 cells transfected with pcDNA3-ckNF45FLAG (DF1/NF45), nontransfected but IBDV-infected DF-1 cells (DF1/D78), and pcDNA3-ckNF45-FLAG-transfected and IBDV-infected Df1 cells (Df1/NF45/D78) were analyzed either directly in a Western blot assay (WB) or were immunoprecipitated (IP) using the anti-FLAG MAb followed by Western blot analysis of the IP products. (B and C) Lysates of the cytoplasmic fractions of noninfected DF-1 cells (DF1) and IBDV-infected DF-1 cells (DF1/D78) were either directly used for WB or for IP using the VP2-specific MAb R63 (B) or the VP3-specific MAb IBDV2 (C) followed by Western blot analysis. Western blots shown in all three panels were performed using rabbit anti-ckNF45-His serum or rabbit anti-VP1, -VP2, or -VP3 serum. The detected proteins are named and marked by arrows. The molecular masses (in kilodaltons) of protein markers are indicated to the left of each gel.

    Techniques Used: Transfection, Infection, Western Blot, Immunoprecipitation

    28) Product Images from "Monovalency Unleashes the Full Therapeutic Potential of the DN-30 Anti-Met Antibody *"

    Article Title: Monovalency Unleashes the Full Therapeutic Potential of the DN-30 Anti-Met Antibody *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.134031

    Engineering, expression, and binding properties of DN-30 Fab. A , schematic representation of the lentiviral vectors encoding DN-30 mAb or Fab. Gray boxes , vector backbone; white boxes , expression cassette. P min CMV , minimal core promoter elements from human cytomegalovirus joined upstream and in opposite orientation to the promoter of the human phosphoglycerate kinase ( PGK ) gene. VL , light chain variable region; CL , light chain constant region; VH , heavy chain variable region; CH1 to - 3 , heavy chain constant regions 1–3; FLAG and HIS , nucleotide sequences encoding for a FLAG epitope and a polyhistidine tail, respectively. B , DN-30 mAb and Fab expression in the conditioned medium of MDA-MB-435 human melanoma cells transduced with the indicated concentrations of p24 equivalent of LV particles. Recombinant proteins were detected by Western blotting under non-reducing conditions using anti-FLAG antibodies. MW , molecular weight. C , ELISA binding analysis of DN-30 mAb and Fab (liquid phase) to a Met-Fc chimera (solid phase). Binding was revealed using anti-FLAG antibodies. Solid square , DN-30 mAb; solid circle , DN-30 Fab; A.U. , arbitrary units. Each point is the mean of triplicate values. Error bars , S.D. D , schematic representation of the DN-30 mAb and Fab proteins. ABS , antigen binding site; Flag , epitope recognized by anti-FLAG antibodies.
    Figure Legend Snippet: Engineering, expression, and binding properties of DN-30 Fab. A , schematic representation of the lentiviral vectors encoding DN-30 mAb or Fab. Gray boxes , vector backbone; white boxes , expression cassette. P min CMV , minimal core promoter elements from human cytomegalovirus joined upstream and in opposite orientation to the promoter of the human phosphoglycerate kinase ( PGK ) gene. VL , light chain variable region; CL , light chain constant region; VH , heavy chain variable region; CH1 to - 3 , heavy chain constant regions 1–3; FLAG and HIS , nucleotide sequences encoding for a FLAG epitope and a polyhistidine tail, respectively. B , DN-30 mAb and Fab expression in the conditioned medium of MDA-MB-435 human melanoma cells transduced with the indicated concentrations of p24 equivalent of LV particles. Recombinant proteins were detected by Western blotting under non-reducing conditions using anti-FLAG antibodies. MW , molecular weight. C , ELISA binding analysis of DN-30 mAb and Fab (liquid phase) to a Met-Fc chimera (solid phase). Binding was revealed using anti-FLAG antibodies. Solid square , DN-30 mAb; solid circle , DN-30 Fab; A.U. , arbitrary units. Each point is the mean of triplicate values. Error bars , S.D. D , schematic representation of the DN-30 mAb and Fab proteins. ABS , antigen binding site; Flag , epitope recognized by anti-FLAG antibodies.

    Techniques Used: Expressing, Binding Assay, Plasmid Preparation, FLAG-tag, Multiple Displacement Amplification, Transduction, Recombinant, Western Blot, Molecular Weight, Enzyme-linked Immunosorbent Assay

    29) Product Images from "JNK-interacting protein-1 promotes transcription of A? protein precursor but not A? precursor-like proteins, mechanistically different than Fe65"

    Article Title: JNK-interacting protein-1 promotes transcription of A? protein precursor but not A? precursor-like proteins, mechanistically different than Fe65

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

    doi: 10.1073/pnas.0437908100

    JIP-1 does not accumulate in the nucleus in the absence of AβPP. ( a ) Flag-tagged JIP-1 was cotransfected with GFP, AβPP-GFP, or AID-GFP. The Flag epitope was located by immunostaining with anti-Flag primary antibody and Cy5 secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Merged images along with the individual channels are presented. Note that the amount of JIP-1 located in the nucleus is nearly the same regardless of which GFP construct is cotransfected. ( b ) GFP-Fe65 was cotransfected with either full-length AβPP or pcDNA3. AβPP was immunostained with anti-AβPPCT primary antibody and Alexa-594 secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole. (Magnifications: ×60.)
    Figure Legend Snippet: JIP-1 does not accumulate in the nucleus in the absence of AβPP. ( a ) Flag-tagged JIP-1 was cotransfected with GFP, AβPP-GFP, or AID-GFP. The Flag epitope was located by immunostaining with anti-Flag primary antibody and Cy5 secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole. Merged images along with the individual channels are presented. Note that the amount of JIP-1 located in the nucleus is nearly the same regardless of which GFP construct is cotransfected. ( b ) GFP-Fe65 was cotransfected with either full-length AβPP or pcDNA3. AβPP was immunostained with anti-AβPPCT primary antibody and Alexa-594 secondary antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole. (Magnifications: ×60.)

    Techniques Used: FLAG-tag, Immunostaining, Staining, Construct

    30) Product Images from "Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein"

    Article Title: Progressive atrioventricular conduction defects and heart failure in mice expressing a mutant Csx/Nkx2.5 homeoprotein

    Journal: Journal of Clinical Investigation

    doi:

    Expression of Csx/Nkx2.5(I183P) and endogenous Csx/Nkx2.5 in TG mice. ( a ) Immunohistochemistry showed Csx/Nkx2.5(I183P) mutant protein expression in atria and ventricle in embryonic, neonatal, and adult atria (#25-A) and ventricles (#25-V). Bars = 50 mm. ( b – g ) Adult heart from NTG ( b , d , f ) and TG ( c , e , g ) mice were coimmunostained with anti-Csx/Nkx2.5 Ab ( d , e , FITC) and anti-FLAG Ab ( f , g , rhodamine). Nuclear staining was shown in Blue ( b , c ). Most of the FITC and rhodamine stainings in e and g were colocalized. Bars = 50 mm. ( h ) Mutant protein expression at 14 (lane 2) and 17 dpc (lane 4) by Western blotting using anti-FLAG pAb (upper panels) and anti-Csx/Nkx2.5 mAb (lower panels). In lane 2 and 4, both endogenous and mutant proteins were recognized with anti-Csx/Nkx2.5 Ab and showed approximately twofold higher Csx/Nkx2.5 protein expression than NTG (lane 1 vs. lane 2, lane 3 vs. lane 4). ( i ) Western blot analysis of heart lysate from neonate, 3 and 6 weeks of NTG and TG mice with anti-Csx/Nkx2.5 mAb detected endogenous protein in NTG hearts (lanes 1, 3, 5) as well as the endogenous plus the mutant protein in TG heart (lanes 2, 4, 6). ( j ) Northern blot analysis of Csx/Nkx2.5 and SV40 poly A. At neonatal stage, Csx/Nkx2.5 mRNA was detected as a major single band in NTG heart (Csx/Nkx2.5, lane 1), and two bands in TG heart (lane 2). The slower migrating ban hybridized with SV40 poly A probe indicating the transcript of I183P mutant (SV40 pA, lane 2). In NTG hearts, Csx/Nkx2.5 mRNA level was downregulated after birth (compare lane 1 vs. lanes 3, 5, 7). However, the downregulation of the endogenous Csx/Nkx2.5 was not observed in TG hearts (lanes 2, 4, 6, 8), indicating the upregulation of endogenous Csx/Nkx2.5 in TG hearts.
    Figure Legend Snippet: Expression of Csx/Nkx2.5(I183P) and endogenous Csx/Nkx2.5 in TG mice. ( a ) Immunohistochemistry showed Csx/Nkx2.5(I183P) mutant protein expression in atria and ventricle in embryonic, neonatal, and adult atria (#25-A) and ventricles (#25-V). Bars = 50 mm. ( b – g ) Adult heart from NTG ( b , d , f ) and TG ( c , e , g ) mice were coimmunostained with anti-Csx/Nkx2.5 Ab ( d , e , FITC) and anti-FLAG Ab ( f , g , rhodamine). Nuclear staining was shown in Blue ( b , c ). Most of the FITC and rhodamine stainings in e and g were colocalized. Bars = 50 mm. ( h ) Mutant protein expression at 14 (lane 2) and 17 dpc (lane 4) by Western blotting using anti-FLAG pAb (upper panels) and anti-Csx/Nkx2.5 mAb (lower panels). In lane 2 and 4, both endogenous and mutant proteins were recognized with anti-Csx/Nkx2.5 Ab and showed approximately twofold higher Csx/Nkx2.5 protein expression than NTG (lane 1 vs. lane 2, lane 3 vs. lane 4). ( i ) Western blot analysis of heart lysate from neonate, 3 and 6 weeks of NTG and TG mice with anti-Csx/Nkx2.5 mAb detected endogenous protein in NTG hearts (lanes 1, 3, 5) as well as the endogenous plus the mutant protein in TG heart (lanes 2, 4, 6). ( j ) Northern blot analysis of Csx/Nkx2.5 and SV40 poly A. At neonatal stage, Csx/Nkx2.5 mRNA was detected as a major single band in NTG heart (Csx/Nkx2.5, lane 1), and two bands in TG heart (lane 2). The slower migrating ban hybridized with SV40 poly A probe indicating the transcript of I183P mutant (SV40 pA, lane 2). In NTG hearts, Csx/Nkx2.5 mRNA level was downregulated after birth (compare lane 1 vs. lanes 3, 5, 7). However, the downregulation of the endogenous Csx/Nkx2.5 was not observed in TG hearts (lanes 2, 4, 6, 8), indicating the upregulation of endogenous Csx/Nkx2.5 in TG hearts.

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Mutagenesis, Staining, Western Blot, Northern Blot

    31) Product Images from "I?B kinase ?-induced phosphorylation of CARMA1 contributes to CARMA1-Bcl10-MALT1 complex formation in B cells"

    Article Title: I?B kinase ?-induced phosphorylation of CARMA1 contributes to CARMA1-Bcl10-MALT1 complex formation in B cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20070379

    Phosphorylation of Ser578 is mediated by IKKβ. (A) The protein expression of IKKβ was analyzed by Western blotting in wild-type or IKKβ-deficient (IKKβ −/− ) DT40 cells. (B) IKK kinase assay in wild-type or IKKβ −/− cells was performed as in Fig. 1 . (C) The phosphorylation status of endogenous CARMA1 in wild-type or IKKβ-deficient (IKKβ −/− ) DT40 cells was determined. Cytosolic extracts from 6 × 10 7 cells per sample were immunoprecipitated with anti-CARMA1 antibody and analyzed by the indicated phospho-specific antibodies. Induced CARMA1 phosphorylation was quantitated as in Fig. 1 C . (D) IKK kinase assay in hemagglutinin-tagged wild-type IKKβ or its S176/181A mutant (SSAA) was performed as in Fig. 1 . Both wild-type and mutant knock-in (KI) cells were generated to transfect each KI construct into IKKβ +/− DT40 cells. (E) Phosphorylation of CARMA1 and recruitment of Bcl10 and MALT1 were examined with Flag-tagged CARMA1 transfected into wild-type– or SSAA-KI cells, as described in Fig. 2 D . Cell lysates (from 3 × 10 7 cells per sample) were immunoprecipitated by anti-Flag mAb and analyzed by Western blotting using the indicated antibodies. The phosphorylated CARMA1 was quantitated as in Fig. 1 C . (F) For in vitro IKKα and β kinase assay, purified Flag-tagged CARMA1 was used as a substrate. Phosphorylated CARMA1 was analyzed by Western blotting with anti–phospho-S578 antibody. WCL, whole cell lysate; wt, wild type.
    Figure Legend Snippet: Phosphorylation of Ser578 is mediated by IKKβ. (A) The protein expression of IKKβ was analyzed by Western blotting in wild-type or IKKβ-deficient (IKKβ −/− ) DT40 cells. (B) IKK kinase assay in wild-type or IKKβ −/− cells was performed as in Fig. 1 . (C) The phosphorylation status of endogenous CARMA1 in wild-type or IKKβ-deficient (IKKβ −/− ) DT40 cells was determined. Cytosolic extracts from 6 × 10 7 cells per sample were immunoprecipitated with anti-CARMA1 antibody and analyzed by the indicated phospho-specific antibodies. Induced CARMA1 phosphorylation was quantitated as in Fig. 1 C . (D) IKK kinase assay in hemagglutinin-tagged wild-type IKKβ or its S176/181A mutant (SSAA) was performed as in Fig. 1 . Both wild-type and mutant knock-in (KI) cells were generated to transfect each KI construct into IKKβ +/− DT40 cells. (E) Phosphorylation of CARMA1 and recruitment of Bcl10 and MALT1 were examined with Flag-tagged CARMA1 transfected into wild-type– or SSAA-KI cells, as described in Fig. 2 D . Cell lysates (from 3 × 10 7 cells per sample) were immunoprecipitated by anti-Flag mAb and analyzed by Western blotting using the indicated antibodies. The phosphorylated CARMA1 was quantitated as in Fig. 1 C . (F) For in vitro IKKα and β kinase assay, purified Flag-tagged CARMA1 was used as a substrate. Phosphorylated CARMA1 was analyzed by Western blotting with anti–phospho-S578 antibody. WCL, whole cell lysate; wt, wild type.

    Techniques Used: Expressing, Western Blot, Kinase Assay, Immunoprecipitation, Mutagenesis, Knock-In, Generated, Construct, Transfection, In Vitro, Purification

    Multiple Ser/Thr residues of CARMA1 are important for BCR-mediated IKK activation. (A) Schematic diagram of various CARMA1 mutants. Arrowheads represent the mutated amino acid indicated as T119A (left). (B) BCR-mediated IKK, ERK, and JNK activation in wild-type and CARMA1-deficient (CARMA1 −/− ) DT40 B cells. IKK kinase assay was measured by phosphorylation of GST-IκBα as a substrate and detected by anti–phos-pho-IκBα mAb (left). Phospho-ERK and -JNK were analyzed by Western blotting (middle and right). (C) For functional analysis of CARMA1 mutants, IKK kinase assay was performed as in B. Wild-type and mutated Flag-tagged CARMA1 cDNAs were transfected into CARMA1 −/− DT40 B cells. Induced IKK activity was quantitated with Multi Gauge software (Fujifilm) and represented as fold activation compared with time zero of the wild type. (top) Protein expression of wild-type and mutated CARMA1, detected by Western blotting with anti-Flag mAb (1 × 10 6 cells per lane). (D) For JNK activation, whole-cell lysates (2 × 10 6 cells per lane) were analyzed by Western blotting with anti–phospho-JNK antibody. (E) Sequence aliments of the important Ser/Thr residues between chicken, mouse, and human CARMA1. wt, wild type.
    Figure Legend Snippet: Multiple Ser/Thr residues of CARMA1 are important for BCR-mediated IKK activation. (A) Schematic diagram of various CARMA1 mutants. Arrowheads represent the mutated amino acid indicated as T119A (left). (B) BCR-mediated IKK, ERK, and JNK activation in wild-type and CARMA1-deficient (CARMA1 −/− ) DT40 B cells. IKK kinase assay was measured by phosphorylation of GST-IκBα as a substrate and detected by anti–phos-pho-IκBα mAb (left). Phospho-ERK and -JNK were analyzed by Western blotting (middle and right). (C) For functional analysis of CARMA1 mutants, IKK kinase assay was performed as in B. Wild-type and mutated Flag-tagged CARMA1 cDNAs were transfected into CARMA1 −/− DT40 B cells. Induced IKK activity was quantitated with Multi Gauge software (Fujifilm) and represented as fold activation compared with time zero of the wild type. (top) Protein expression of wild-type and mutated CARMA1, detected by Western blotting with anti-Flag mAb (1 × 10 6 cells per lane). (D) For JNK activation, whole-cell lysates (2 × 10 6 cells per lane) were analyzed by Western blotting with anti–phospho-JNK antibody. (E) Sequence aliments of the important Ser/Thr residues between chicken, mouse, and human CARMA1. wt, wild type.

    Techniques Used: Activation Assay, Kinase Assay, Western Blot, Functional Assay, Transfection, Activity Assay, Software, Expressing, Sequencing

    Thr119, Ser578, and Ser668 on CARMA1 are phosphorylated upon BCR stimulation. (A) Cytosolic extracts (from 2 × 10 7 cells per sample) were immunoprecipitated with anti-Flag mAb and analyzed by Western blotting. The phosphorylated CARMA1 was detected by each phosphospecific antibody (anti-pT119, -pS578, and -pS668). The arrowhead indicates the position of phosphorylated S578 of CARMA1. (B) Phosphorylation status of CARMA1 in wild-type or PKCβ-deficient (PKCβ −/− ) DT40 cells was determined by the same procedures as in A. (C) For in vitro PKCβ kinase assay, purified Flag-tagged CARMA1 protein was used as a substrate. Phosphorylated CARMA1 was analyzed by Western blotting with anti–phospho-S668 antibody. (D) For association of Bcl10 or MALT1 with CARMA1, wild-type and mutated Flag-tagged CARMA1 cDNAs were transfected with pBIG vectors, as described in Materials and methods, into CARMA1 −/− DT40 B cells. Cell lysates (from 3 × 10 7 cells per sample) were immunoprecipitated by anti-Flag mAb and analyzed by Western blotting using anti-Bcl10 mAb or anti-MALT1 antibody. WCL, whole cell lysate; wt, wild type.
    Figure Legend Snippet: Thr119, Ser578, and Ser668 on CARMA1 are phosphorylated upon BCR stimulation. (A) Cytosolic extracts (from 2 × 10 7 cells per sample) were immunoprecipitated with anti-Flag mAb and analyzed by Western blotting. The phosphorylated CARMA1 was detected by each phosphospecific antibody (anti-pT119, -pS578, and -pS668). The arrowhead indicates the position of phosphorylated S578 of CARMA1. (B) Phosphorylation status of CARMA1 in wild-type or PKCβ-deficient (PKCβ −/− ) DT40 cells was determined by the same procedures as in A. (C) For in vitro PKCβ kinase assay, purified Flag-tagged CARMA1 protein was used as a substrate. Phosphorylated CARMA1 was analyzed by Western blotting with anti–phospho-S668 antibody. (D) For association of Bcl10 or MALT1 with CARMA1, wild-type and mutated Flag-tagged CARMA1 cDNAs were transfected with pBIG vectors, as described in Materials and methods, into CARMA1 −/− DT40 B cells. Cell lysates (from 3 × 10 7 cells per sample) were immunoprecipitated by anti-Flag mAb and analyzed by Western blotting using anti-Bcl10 mAb or anti-MALT1 antibody. WCL, whole cell lysate; wt, wild type.

    Techniques Used: Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, Purification, Transfection

    32) Product Images from "HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin"

    Article Title: HIV-1 integrase modulates the interaction of the HIV-1 cellular cofactor LEDGF/p75 with chromatin

    Journal: Retrovirology

    doi: 10.1186/1742-4690-8-27

    Effect of HIV-1 integrase Q168L mutant on the chromatin binding activity of LEDGF/p75 . Immunoblots show the effect of transient expression of HIV integrase WT and Q168L mutant on the chromatin binding strength of LEDGF/p75 WT (a) and LEDGF/p75 ΔPWWP (b), and the effect of HIV-1 integrase WT on the chromatin binding strength of LEDGF/p75 ΔIBD (c). LEDGF/p75 was detected with an anti-FLAG Mab. Immunoblots in (d) show the level of expression of HIV integrase WT and Q168L in cells coexpressing LEDGF/p75 WT, LEDGF/p75 ΔPWWP, and LEDGF/p75 ΔIBD analyzed in the experiments represented in (a), (b), and (c), respectively. HIV-1 Integrase was detected with an anti-Myc Mab. T represents a total cellular lysate.
    Figure Legend Snippet: Effect of HIV-1 integrase Q168L mutant on the chromatin binding activity of LEDGF/p75 . Immunoblots show the effect of transient expression of HIV integrase WT and Q168L mutant on the chromatin binding strength of LEDGF/p75 WT (a) and LEDGF/p75 ΔPWWP (b), and the effect of HIV-1 integrase WT on the chromatin binding strength of LEDGF/p75 ΔIBD (c). LEDGF/p75 was detected with an anti-FLAG Mab. Immunoblots in (d) show the level of expression of HIV integrase WT and Q168L in cells coexpressing LEDGF/p75 WT, LEDGF/p75 ΔPWWP, and LEDGF/p75 ΔIBD analyzed in the experiments represented in (a), (b), and (c), respectively. HIV-1 Integrase was detected with an anti-Myc Mab. T represents a total cellular lysate.

    Techniques Used: Mutagenesis, Binding Assay, Activity Assay, Western Blot, Expressing

    HIV-1 integrase promotes binding of LEDGF/p75 ΔPWWP to mitotic chromosomes . (a) Immunofluorescence analysis of LEDGF/p75-deficient cells stably expressing eGFP-tagged HIV-1 integrase and transiently transfected with FLAG-tagged LEDGF/p75 WT (panel ii) or ΔPWWP (panel i). LEDGF/p75 was detected with an anti-FLAG Mab, DAPI was used for detection of chromatin. (b) Integrase-to-chromatin tethering assay. LEDGF/p75-deficient HEK293T cells stably expressing eGFP-tagged integrase were transiently transfected with LEDGF/p75 WT (panel i) or the mutants ΔIBD (panel ii) and ΔPWWP (panel iii) and the subcellular distribution of eGFP-integrase determined by fluorescence microscopy analysis.
    Figure Legend Snippet: HIV-1 integrase promotes binding of LEDGF/p75 ΔPWWP to mitotic chromosomes . (a) Immunofluorescence analysis of LEDGF/p75-deficient cells stably expressing eGFP-tagged HIV-1 integrase and transiently transfected with FLAG-tagged LEDGF/p75 WT (panel ii) or ΔPWWP (panel i). LEDGF/p75 was detected with an anti-FLAG Mab, DAPI was used for detection of chromatin. (b) Integrase-to-chromatin tethering assay. LEDGF/p75-deficient HEK293T cells stably expressing eGFP-tagged integrase were transiently transfected with LEDGF/p75 WT (panel i) or the mutants ΔIBD (panel ii) and ΔPWWP (panel iii) and the subcellular distribution of eGFP-integrase determined by fluorescence microscopy analysis.

    Techniques Used: Binding Assay, Immunofluorescence, Stable Transfection, Expressing, Transfection, Fluorescence, Microscopy

    HIV-1 cofactor activity of LEDGF/p75 ΔPWWP . (a) T L3 , and T L3 cells expressing LEDGF/p75 WT or LEDGF/p75 ΔPWWP (eleven different cell lines) were challenged with HIVluc and luciferase activity determined five days later. Expression of LEDGF/p75 proteins in these cell lines was documented by immunoblotting with an anti-LEDGF and anti-FLAG Mabs. Errors bars correspond to four independent infection experiments. (b) LEDGF/p75 mRNA levels in T C3 and cells used in panel (a). The mRNA levels of endogenous LEDGF/p75 were quantified by real time PCR using specific primers. mRNA levels for LEDGF/p75 were normalized to those of GAPDH in the same samples. Errors bars correspond to triplicate real-time PCR measurements.
    Figure Legend Snippet: HIV-1 cofactor activity of LEDGF/p75 ΔPWWP . (a) T L3 , and T L3 cells expressing LEDGF/p75 WT or LEDGF/p75 ΔPWWP (eleven different cell lines) were challenged with HIVluc and luciferase activity determined five days later. Expression of LEDGF/p75 proteins in these cell lines was documented by immunoblotting with an anti-LEDGF and anti-FLAG Mabs. Errors bars correspond to four independent infection experiments. (b) LEDGF/p75 mRNA levels in T C3 and cells used in panel (a). The mRNA levels of endogenous LEDGF/p75 were quantified by real time PCR using specific primers. mRNA levels for LEDGF/p75 were normalized to those of GAPDH in the same samples. Errors bars correspond to triplicate real-time PCR measurements.

    Techniques Used: Activity Assay, Expressing, Luciferase, Infection, Real-time Polymerase Chain Reaction

    33) Product Images from "Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease"

    Article Title: Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease

    Journal: Journal of Clinical Investigation

    doi:

    Expression of translated products in cells: intron-splicing defect in M112 mutant resulted in poor protein accumulation. ( a ) Wild-type and mutant CSX/NKX2.5 expression vectors were transfected into COS 7 cells, and the protein expression was examined by Western blotting using anti-FLAG Ab approximately 24 hours after transfection (FLAG, top). All mutant proteins except M112 (lane 11, asterisk indicates the expected molecular weight of M112 protein) were detected at the expected molecular weight. GAPDH expression in each lane was also shown (GAPDH, bottom). ( b ) Wild-type and mutant CSX/NKX2.5 proteins accumulated in the nucleus colocalizing with Hoechst nuclear staining (NUC, lower panels). The results presented are wild-type, group 1 (M170), group 2 (M191), group 3 (M198), and group 4 (M25). ( c ) G→T substitution (large arrow) identified in M112 mutant on the CSX/NKX2.5 splicing donor site was examined by RT-PCR. RNA-purified form COS 7 cells transfected with the wild-type and M112 mutant of CSX/NKX2.5 were amplified with two primers spanning the intron. In the wild-type transfectant, the intron was spliced out, resulting in the generation of 240-bp product, whereas in the mutant transfectant, G→T substitution of the first codon of the intron splicing site abolished the normal splicing, resulting in generation of 1,779-bp product. CSX/NKX2.5 protein was encoded by the two exons represented. ( d ) Translated products were examined approximately 24 hours after transfection by Western blotting using anti-FLAG mAb (top) and anti-Csx/Nkx2.5 mAb (bottom). Wild-type CSX/NKX2.5 gene was translated into approximately 42-kDa protein and was detected with anti-FLAG and anti-Csx/Nkx2.5 mAb (lane 1). In contrast, M112 mutant protein that is expected to migrate approximately 29 kDa was not detected in the cell lysate (lane 2). In in vitro transcription and translation system, cDNA produced approximately 42-kDa protein (lane 1), and M112 genomic construct produced about 29-kDa protein (lane 2).
    Figure Legend Snippet: Expression of translated products in cells: intron-splicing defect in M112 mutant resulted in poor protein accumulation. ( a ) Wild-type and mutant CSX/NKX2.5 expression vectors were transfected into COS 7 cells, and the protein expression was examined by Western blotting using anti-FLAG Ab approximately 24 hours after transfection (FLAG, top). All mutant proteins except M112 (lane 11, asterisk indicates the expected molecular weight of M112 protein) were detected at the expected molecular weight. GAPDH expression in each lane was also shown (GAPDH, bottom). ( b ) Wild-type and mutant CSX/NKX2.5 proteins accumulated in the nucleus colocalizing with Hoechst nuclear staining (NUC, lower panels). The results presented are wild-type, group 1 (M170), group 2 (M191), group 3 (M198), and group 4 (M25). ( c ) G→T substitution (large arrow) identified in M112 mutant on the CSX/NKX2.5 splicing donor site was examined by RT-PCR. RNA-purified form COS 7 cells transfected with the wild-type and M112 mutant of CSX/NKX2.5 were amplified with two primers spanning the intron. In the wild-type transfectant, the intron was spliced out, resulting in the generation of 240-bp product, whereas in the mutant transfectant, G→T substitution of the first codon of the intron splicing site abolished the normal splicing, resulting in generation of 1,779-bp product. CSX/NKX2.5 protein was encoded by the two exons represented. ( d ) Translated products were examined approximately 24 hours after transfection by Western blotting using anti-FLAG mAb (top) and anti-Csx/Nkx2.5 mAb (bottom). Wild-type CSX/NKX2.5 gene was translated into approximately 42-kDa protein and was detected with anti-FLAG and anti-Csx/Nkx2.5 mAb (lane 1). In contrast, M112 mutant protein that is expected to migrate approximately 29 kDa was not detected in the cell lysate (lane 2). In in vitro transcription and translation system, cDNA produced approximately 42-kDa protein (lane 1), and M112 genomic construct produced about 29-kDa protein (lane 2).

    Techniques Used: Expressing, Mutagenesis, Transfection, Western Blot, Molecular Weight, Staining, Reverse Transcription Polymerase Chain Reaction, Purification, Amplification, In Vitro, Produced, Construct

    34) Product Images from "Dynamic Modification of Sphingomyelin in Lipid Microdomains Controls Development of Obesity, Fatty Liver, and Type 2 Diabetes *"

    Article Title: Dynamic Modification of Sphingomyelin in Lipid Microdomains Controls Development of Obesity, Fatty Liver, and Type 2 Diabetes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.255646

    SMS2 associates with CD36/FAT and caveolin 1 and is involved in lipid droplet formation. A, FLAG-tagged SMS2 and V5-tagged CD36/FAT were co-expressed in COS7 cells, and DIM fractions were prepared, and then each protein was detected by Western blotting.
    Figure Legend Snippet: SMS2 associates with CD36/FAT and caveolin 1 and is involved in lipid droplet formation. A, FLAG-tagged SMS2 and V5-tagged CD36/FAT were co-expressed in COS7 cells, and DIM fractions were prepared, and then each protein was detected by Western blotting.

    Techniques Used: Western Blot

    35) Product Images from "Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore"

    Article Title: Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200409149

    Rapamycin-induced accumulation of reporter protein at the NE. HeLa cells were cotransfected with the reporter and trap plasmids, and after 20 h were incubated with or without 150 ng/ml rapamycin (rap) at 37°C for 2 h. Cells were washed in buffer lacking or containing 1% Triton X-100 and fixed. The reporter protein was visualized by GFP fluorescence (green), whereas the trap protein was visualized by immunofluorescent staining with anti-FLAG mAb (red). Similar results were obtained upon treatment of cells with rapamycin for 30 min instead of 2 h (not depicted).
    Figure Legend Snippet: Rapamycin-induced accumulation of reporter protein at the NE. HeLa cells were cotransfected with the reporter and trap plasmids, and after 20 h were incubated with or without 150 ng/ml rapamycin (rap) at 37°C for 2 h. Cells were washed in buffer lacking or containing 1% Triton X-100 and fixed. The reporter protein was visualized by GFP fluorescence (green), whereas the trap protein was visualized by immunofluorescent staining with anti-FLAG mAb (red). Similar results were obtained upon treatment of cells with rapamycin for 30 min instead of 2 h (not depicted).

    Techniques Used: Incubation, Fluorescence, Staining

    36) Product Images from "Implication of Serine Residues 271, 273, and 275 in the Human Immunodeficiency Virus Type 1 Cofactor Activity of Lens Epithelium-Derived Growth Factor/p75 ▿"

    Article Title: Implication of Serine Residues 271, 273, and 275 in the Human Immunodeficiency Virus Type 1 Cofactor Activity of Lens Epithelium-Derived Growth Factor/p75 ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01043-09

    Interaction of LEDGF/p75 mutants with HIV-1 integrase. (a) Integrase protection assay. LEDGF/p75-deficient HEK293T cells stably expressing Myc-tagged HIV-1 integrase were either transiently transfected or not transfected with plasmids expressing the LEDGF/p75 WT (I) or mutants (II). Expression levels of LEDGF/p75 proteins and HIV-1 integrase were determined by immunoblotting with an anti-FLAG and anti-Myc MAbs, respectively. Detection of endogenous c-Myc was used as a loading control for these experiments. (b) Coimmunoprecipitation of HIV-1 integrase with the LEDGF/p75 ΔCR3 mutant. LEDGF/p75-deficient HEK293T cells were cotransfected with the FLAG-tagged LEDGF/p75 WT or different mutants and Myc-tagged HIV-1 and subjected to immunoprecipitation with an anti-FLAG MAb. Immunoprecipitated proteins were detected by immunoblotting with anti-Myc or anti-FLAG MAbs. Mouse antibody light chains were used as a loading control for the immunoprecipitation.
    Figure Legend Snippet: Interaction of LEDGF/p75 mutants with HIV-1 integrase. (a) Integrase protection assay. LEDGF/p75-deficient HEK293T cells stably expressing Myc-tagged HIV-1 integrase were either transiently transfected or not transfected with plasmids expressing the LEDGF/p75 WT (I) or mutants (II). Expression levels of LEDGF/p75 proteins and HIV-1 integrase were determined by immunoblotting with an anti-FLAG and anti-Myc MAbs, respectively. Detection of endogenous c-Myc was used as a loading control for these experiments. (b) Coimmunoprecipitation of HIV-1 integrase with the LEDGF/p75 ΔCR3 mutant. LEDGF/p75-deficient HEK293T cells were cotransfected with the FLAG-tagged LEDGF/p75 WT or different mutants and Myc-tagged HIV-1 and subjected to immunoprecipitation with an anti-FLAG MAb. Immunoprecipitated proteins were detected by immunoblotting with anti-Myc or anti-FLAG MAbs. Mouse antibody light chains were used as a loading control for the immunoprecipitation.

    Techniques Used: Stable Transfection, Expressing, Transfection, Mutagenesis, Immunoprecipitation

    Chromatin-binding activity of LEDGF/p75 mutants. (a) Chromatin-binding assay. T L3 cells expressing different LEDGF/p75 mutants were fractionated into non-chromatin-bound fractions (S1 and P2) and chromatin-bound fractions (P1 and S2), and the presence of LEDGF/p75 in these fractions was evaluated by immunoblotting with an anti-FLAG MAb. An unfractionated total cellular fraction (T) was included as a control. (b) Salt extraction analysis of LEDGF/p75 mutants stably expressed in T L3 cells. The salt concentration that extracted the LEDGF/p75 WT from chromatin is marked with a rectangle.
    Figure Legend Snippet: Chromatin-binding activity of LEDGF/p75 mutants. (a) Chromatin-binding assay. T L3 cells expressing different LEDGF/p75 mutants were fractionated into non-chromatin-bound fractions (S1 and P2) and chromatin-bound fractions (P1 and S2), and the presence of LEDGF/p75 in these fractions was evaluated by immunoblotting with an anti-FLAG MAb. An unfractionated total cellular fraction (T) was included as a control. (b) Salt extraction analysis of LEDGF/p75 mutants stably expressed in T L3 cells. The salt concentration that extracted the LEDGF/p75 WT from chromatin is marked with a rectangle.

    Techniques Used: Binding Assay, Activity Assay, Expressing, Stable Transfection, Concentration Assay

    Evaluation of the HIV-1 cofactor activities of LEDGF/p75 deletion mutants. (a) Schematic representation of deleted regions. (b) Different LEDGF/p75 deletion mutants were stably expressed in LEDGF/p75 knockdown T L3 cells, and their protein levels were evaluated by immunoblotting using anti-LEDGF or anti-FLAG MAbs. Alpha-tubulin detection was used as a loading control. P and P/A represents PWWP and PWWP/AT hook, respectively. (c) Single-round HIV-1 infections of T L3 -derived cells. Cells immunoblotted in b were challenged with HIVluc, and luciferase activity was determined 5 days later.
    Figure Legend Snippet: Evaluation of the HIV-1 cofactor activities of LEDGF/p75 deletion mutants. (a) Schematic representation of deleted regions. (b) Different LEDGF/p75 deletion mutants were stably expressed in LEDGF/p75 knockdown T L3 cells, and their protein levels were evaluated by immunoblotting using anti-LEDGF or anti-FLAG MAbs. Alpha-tubulin detection was used as a loading control. P and P/A represents PWWP and PWWP/AT hook, respectively. (c) Single-round HIV-1 infections of T L3 -derived cells. Cells immunoblotted in b were challenged with HIVluc, and luciferase activity was determined 5 days later.

    Techniques Used: Stable Transfection, Derivative Assay, Luciferase, Activity Assay

    (a) Salt extraction assay. T L3 cells expressing the LEDGF/p75 WT or WT-FLAG were lysed in a buffer containing increasing NaCl concentrations. Cells were separated into a soluble fraction and an insoluble fraction by centrifugation, and the presence of LEDGF/p75 was evaluated by immunoblotting with an anti-LEDGF or anti-FLAG MAb, as indicated. An unfractionated total cellular fraction (T) was included as a control. (b) Integrase protection assay. LEDGF/p75-deficient HEK293T cells stably expressing Myc-tagged HIV-1 integrase were either transiently transfected or not transfected with plasmids expressing the LEDGF/p75 WT, WT-FLAG, or ΔIBD-FLAG. Expression levels of LEDGF/p75 proteins and HIV-1 integrase were determined by immunoblotting with anti-LEDGF and anti-Myc MAbs, respectively. (c) Coimmunoprecipitation of HIV-1 integrase with LEDGF/p75. LEDGF/p75-deficient HEK293T cells were cotransfected with plasmids expressing the LEDGF/p75 WT, WT-FLAG, or ΔIBD-FLAG and Myc-tagged HIV-1 integrase and subjected to immunoprecipitation with an anti-LEDGF MAb. Immunoprecipitated proteins were detected by immunoblotting with anti-Myc or anti-LEDGF MAbs. Mouse antibody heavy and light chains were used as a loading control for the immunoprecipitation. LEDGF/p75 and HIV integrase proteins were detected in the samples used for immunoprecipitation (input) by immunoblotting with anti-LEDGF and anti-Myc MAbs, respectively. (d) Single-round infection of T L3 cells expressing the LEDGF/p75 WT or WT-FLAG. Cells were challenged with HIVluc, and luciferase activity was analyzed 5 days later. Luciferase levels detected in T L3 cells expressing the LEDGF/p75 WT were considered to be 100%. Error bars indicate standard deviation values calculated for a number ( n ) of independent infection experiments performed on different days with different viral preparations.
    Figure Legend Snippet: (a) Salt extraction assay. T L3 cells expressing the LEDGF/p75 WT or WT-FLAG were lysed in a buffer containing increasing NaCl concentrations. Cells were separated into a soluble fraction and an insoluble fraction by centrifugation, and the presence of LEDGF/p75 was evaluated by immunoblotting with an anti-LEDGF or anti-FLAG MAb, as indicated. An unfractionated total cellular fraction (T) was included as a control. (b) Integrase protection assay. LEDGF/p75-deficient HEK293T cells stably expressing Myc-tagged HIV-1 integrase were either transiently transfected or not transfected with plasmids expressing the LEDGF/p75 WT, WT-FLAG, or ΔIBD-FLAG. Expression levels of LEDGF/p75 proteins and HIV-1 integrase were determined by immunoblotting with anti-LEDGF and anti-Myc MAbs, respectively. (c) Coimmunoprecipitation of HIV-1 integrase with LEDGF/p75. LEDGF/p75-deficient HEK293T cells were cotransfected with plasmids expressing the LEDGF/p75 WT, WT-FLAG, or ΔIBD-FLAG and Myc-tagged HIV-1 integrase and subjected to immunoprecipitation with an anti-LEDGF MAb. Immunoprecipitated proteins were detected by immunoblotting with anti-Myc or anti-LEDGF MAbs. Mouse antibody heavy and light chains were used as a loading control for the immunoprecipitation. LEDGF/p75 and HIV integrase proteins were detected in the samples used for immunoprecipitation (input) by immunoblotting with anti-LEDGF and anti-Myc MAbs, respectively. (d) Single-round infection of T L3 cells expressing the LEDGF/p75 WT or WT-FLAG. Cells were challenged with HIVluc, and luciferase activity was analyzed 5 days later. Luciferase levels detected in T L3 cells expressing the LEDGF/p75 WT were considered to be 100%. Error bars indicate standard deviation values calculated for a number ( n ) of independent infection experiments performed on different days with different viral preparations.

    Techniques Used: Expressing, Centrifugation, Stable Transfection, Transfection, Immunoprecipitation, Infection, Luciferase, Activity Assay, Standard Deviation

    Related Articles

    Immunoprecipitation:

    Article Title: Bone-Specific Transcription Factor Runx2 Interacts with the 1?,25-Dihydroxyvitamin D3 Receptor To Up-Regulate Rat Osteocalcin Gene Expression in Osteoblastic Cells
    Article Snippet: .. VDR was immunoprecipitated with an anti-VDR monoclonal antibody (Oncogene Research, Boston, Mass.), and the coimmunoprecipitated Runx2 was detected by Western blotting with an anti-Runx2 polyclonal antibody (Santa Cruz Biotechnology). ..

    Methylation:

    Article Title: Characterization of sub-nuclear changes in Caenorhabditis elegans embryos exposed to brief, intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest
    Article Snippet: .. The following primary antibodies were used for these studies: rabbit anti-Phos H3 to detect the phosphorylated (Ser10) form of histone H3 (Update Biotechnology, Lake Placid, NY); mAb414 to detect nuclear envelope pore complexes (BabCo, Richmond, CA), mouse anti-SAN-1 to detect SAN-1 [ ]; mAb DM1A (Sigma-Aldrich, St. Louis, MO) YL1/2 (AbCam, Cambridge MA) to detect alpha tubulin, MetH3 to detect the methylated form of Histone H3 (BabCo, Richmond, CA), anti-gamma tubulin to detect the centrosome (Sigma-Aldrich, St. Louis, MO). ..

    Blocking Assay:

    Article Title: Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray
    Article Snippet: .. After blocking with 5% nonfat milk in PBS overnight at 4°C, the membrane was incubated with MAb D7 (diluted 1:2,000 in PBS) at 37°C for 1 h, washed three times with PBS containing 0.05% (w/v) Tween 20 (PBST, pH 7.4), and probed with a 1:5,000 dilution of HRP-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO, USA) at 37°C for 1 h. Reactivity was visualized with the substrate 3, 3'-diaminobenzidine (DAB, Sigma). .. Indirect immunofluorescence assay The NS1 gene was amplified using primer pairs NS1-pU (5′- ACACGAGCTCATGGATTCCAACACTGTG-3′) and NS1-pL (5′- CCGCTCGAGTCAAACTTCTGACTCAATTG-′3) from the pET-30a-NS1 and cloned into vector pCAGGS with chicken β-actin/rabbit β-globin hybrid promoter (AG) and the human CMV-IE enhancer in various mammalian cells.

    FLAG-tag:

    Article Title: LACE1 interacts with p53 and mediates its mitochondrial translocation and apoptosis
    Article Snippet: .. The M2 monoclonal antibody to FLAG epitope was obtained from Sigma Aldrich, USA. .. Antibody to mtHSP70 was from Lonza, Switzerland and antibodies to p53, Cl-PARP and α-tubulin were from Cell Signaling Technology, USA.

    Incubation:

    Article Title: Single Domain Intracellular Antibodies from Diverse Libraries
    Article Snippet: .. To detect protein expression in the cells, the harvested cells were lysed by resuspending in radioimmune precipitation assay buffer (50 m m Tris, pH 8.0, 1% Nonidet P-40, 150 m m NaCl, 1% sodium deoxycholate, and 0.1% SDS) and incubated on ice for 30 min. After spinning, the supernatants were fractionated by 15% SDS-PAGE gel, transferred to PVDF membranes, and were immunodetected with anti-LMO2 monoclonal antibody ( ) and anti-α-tubulin monoclonal antibody (B-5-1-2, Sigma) as the SDS-PAGE loading control. .. A transgenic line has been established in which Lmo2 is expressed under the control of the T-cell promoter Lck and transplantable T-cell neoplasias arise in these mice, manifest by thymoma and splenomegaly.

    Article Title: Identification of a Highly Conserved Epitope on Avian Influenza Virus Non-Structural Protein 1 Using a Peptide Microarray
    Article Snippet: .. After blocking with 5% nonfat milk in PBS overnight at 4°C, the membrane was incubated with MAb D7 (diluted 1:2,000 in PBS) at 37°C for 1 h, washed three times with PBS containing 0.05% (w/v) Tween 20 (PBST, pH 7.4), and probed with a 1:5,000 dilution of HRP-conjugated goat anti-mouse IgG (Sigma, St. Louis, MO, USA) at 37°C for 1 h. Reactivity was visualized with the substrate 3, 3'-diaminobenzidine (DAB, Sigma). .. Indirect immunofluorescence assay The NS1 gene was amplified using primer pairs NS1-pU (5′- ACACGAGCTCATGGATTCCAACACTGTG-3′) and NS1-pL (5′- CCGCTCGAGTCAAACTTCTGACTCAATTG-′3) from the pET-30a-NS1 and cloned into vector pCAGGS with chicken β-actin/rabbit β-globin hybrid promoter (AG) and the human CMV-IE enhancer in various mammalian cells.

    Article Title: Osteopontin is a tumor autoantigen in prostate cancer patients
    Article Snippet: .. Assays with OPN monoclonal antibody were performed in identical conditions to those described above, using the OPN mAb (Chemicon, Canada, USA) in a 1:2,500 dilution followed by incubation in a 1:5,000 dilution of anti-rat IgG conjugated to horseradish peroxidase (HRP). .. Immunoreactive complexes were developed with the ECL Plus System (Amersham Biosciences) after exposure to Kodak BioMax Light Film.

    Expressing:

    Article Title: Single Domain Intracellular Antibodies from Diverse Libraries
    Article Snippet: .. To detect protein expression in the cells, the harvested cells were lysed by resuspending in radioimmune precipitation assay buffer (50 m m Tris, pH 8.0, 1% Nonidet P-40, 150 m m NaCl, 1% sodium deoxycholate, and 0.1% SDS) and incubated on ice for 30 min. After spinning, the supernatants were fractionated by 15% SDS-PAGE gel, transferred to PVDF membranes, and were immunodetected with anti-LMO2 monoclonal antibody ( ) and anti-α-tubulin monoclonal antibody (B-5-1-2, Sigma) as the SDS-PAGE loading control. .. A transgenic line has been established in which Lmo2 is expressed under the control of the T-cell promoter Lck and transplantable T-cell neoplasias arise in these mice, manifest by thymoma and splenomegaly.

    Western Blot:

    Article Title: Bone-Specific Transcription Factor Runx2 Interacts with the 1?,25-Dihydroxyvitamin D3 Receptor To Up-Regulate Rat Osteocalcin Gene Expression in Osteoblastic Cells
    Article Snippet: .. VDR was immunoprecipitated with an anti-VDR monoclonal antibody (Oncogene Research, Boston, Mass.), and the coimmunoprecipitated Runx2 was detected by Western blotting with an anti-Runx2 polyclonal antibody (Santa Cruz Biotechnology). ..

    SDS Page:

    Article Title: Single Domain Intracellular Antibodies from Diverse Libraries
    Article Snippet: .. To detect protein expression in the cells, the harvested cells were lysed by resuspending in radioimmune precipitation assay buffer (50 m m Tris, pH 8.0, 1% Nonidet P-40, 150 m m NaCl, 1% sodium deoxycholate, and 0.1% SDS) and incubated on ice for 30 min. After spinning, the supernatants were fractionated by 15% SDS-PAGE gel, transferred to PVDF membranes, and were immunodetected with anti-LMO2 monoclonal antibody ( ) and anti-α-tubulin monoclonal antibody (B-5-1-2, Sigma) as the SDS-PAGE loading control. .. A transgenic line has been established in which Lmo2 is expressed under the control of the T-cell promoter Lck and transplantable T-cell neoplasias arise in these mice, manifest by thymoma and splenomegaly.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Millipore monoclonal anti flag m2 antibody
    V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an <t>anti-c-Myc</t> antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with <t>anti-FLAG</t> antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.
    Monoclonal Anti Flag M2 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2 antibody/product/Millipore
    Average 99 stars, based on 796 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag m2 antibody - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an anti-c-Myc antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with anti-FLAG antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: V H H3-sIgA subunits physically associate with one another. N. benthamiana leaf tissue co-infiltrated with all V H H3-sIgA subunits was collected at 6 dpi, and recombinant proteins were immunoprecipitated with an anti-c-Myc antibody. (A,B) Both cell extracts and immunoprecipitates were resolved by SDS-PAGE under reducing conditions. (C,D) SDS-PAGE performed under non-reducing conditions. (A,C) Immunoblots were detected with anti-FLAG antibodies. (B,D) Immunoblots were detected with anti-HA antibodies. Arrow points to a faint but nonetheless present monomeric JC band. TSP from p19-infiltrated N. benthamiana leaves was used as negative control.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Recombinant, Immunoprecipitation, SDS Page, Western Blot, Negative Control

    Vacuum infiltration and purification of V H H9-sIgA. N. benthamiana leaves were vacuum infiltrated with a mixture of V H H9-Fc/SC/JC and p19. Tissue was collected at 12 dpi. Cell extracts were prepared under native conditions and separated with SDS-PAGE under non-reducing conditions. (A) Secretory IgA was purified with peptide M Agarose. Western blots were detected with anti-c-Myc antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1), tetrameric (No. 2, ∼176 kDa), trimeric (No. 3, ∼132 kDa), dimeric (No. 4, ∼88 kDa), and monomeric (No. 5, ∼44 kDa) V H H9-Fc. (B) Secretory IgA was purified with anti-FLAG agarose. Western blots were detected with anti-Flag antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1, ∼66 kDa), SC/trimeric V H H9-Fc/JC (No. 2, ∼206 kDa), SC/dimeric V H H9-Fc (No. 3, ∼160), SC/monomeric V H H9-Fc (No. 4, ∼110 kDa), and monomeric SC (No. 5). 10 μl of cell extract was loaded as a snapshot of the antibody produced in vivo .

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Vacuum infiltration and purification of V H H9-sIgA. N. benthamiana leaves were vacuum infiltrated with a mixture of V H H9-Fc/SC/JC and p19. Tissue was collected at 12 dpi. Cell extracts were prepared under native conditions and separated with SDS-PAGE under non-reducing conditions. (A) Secretory IgA was purified with peptide M Agarose. Western blots were detected with anti-c-Myc antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1), tetrameric (No. 2, ∼176 kDa), trimeric (No. 3, ∼132 kDa), dimeric (No. 4, ∼88 kDa), and monomeric (No. 5, ∼44 kDa) V H H9-Fc. (B) Secretory IgA was purified with anti-FLAG agarose. Western blots were detected with anti-Flag antibody. Arrows indicate the expected size of fully assembled sIgA (No. 1, ∼66 kDa), SC/trimeric V H H9-Fc/JC (No. 2, ∼206 kDa), SC/dimeric V H H9-Fc (No. 3, ∼160), SC/monomeric V H H9-Fc (No. 4, ∼110 kDa), and monomeric SC (No. 5). 10 μl of cell extract was loaded as a snapshot of the antibody produced in vivo .

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Purification, SDS Page, Western Blot, Produced, In Vivo

    Binding of plant-produced V H H9-sIgA to EHEC O157:H7 intimin. (A) SPR binding of V H H9-sIgA purified using peptide M. Either plant-produced V H H9-sIgA (top) or E. coli -produced V H H9 monomer (bottom) was immobilized on CM5 Series S sensor chips via amine coupling and MBP-Int277 was flowed over the resulting surfaces at concentrations ranging from 0.3 to 5 nM. The experiment was conducted in duplicate. Black lines show data and red lines show fits. (B) ELISA binding of plant-produced V H H9-sIgA purified using either peptide M (left) or anti-FLAG antibody (right) and detected using either anti-bovine IgA antibody (top) or anti-FLAG antibody (bottom). Results are representative of two independent experiments.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Binding of plant-produced V H H9-sIgA to EHEC O157:H7 intimin. (A) SPR binding of V H H9-sIgA purified using peptide M. Either plant-produced V H H9-sIgA (top) or E. coli -produced V H H9 monomer (bottom) was immobilized on CM5 Series S sensor chips via amine coupling and MBP-Int277 was flowed over the resulting surfaces at concentrations ranging from 0.3 to 5 nM. The experiment was conducted in duplicate. Black lines show data and red lines show fits. (B) ELISA binding of plant-produced V H H9-sIgA purified using either peptide M (left) or anti-FLAG antibody (right) and detected using either anti-bovine IgA antibody (top) or anti-FLAG antibody (bottom). Results are representative of two independent experiments.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Binding Assay, Produced, SPR Assay, Purification, Enzyme-linked Immunosorbent Assay

    Design and production of individual subunits required for chimeric secretory IgA assembly. (A) Schematic of all produced subunits fully assembled into a chimeric antibody intended for secretory IgA functionality. It notably differs from the structure of native secretory IgA by the replacement of the Fab region with a camelid-derived variable heavy chain fragment (V H H). (B) Schematic representation of constructs used for Agrobacterium -mediated transient expression in N. benthamiana leaves. CaMV 35S, cauliflower mosaic virus 35S promoter; CPMV 5′UTR, 5′-untranslated region of Cowpea mosaic virus; PR1b, tobacco pathogenesis-related protein 1b signal peptide; V H Hx-Fc, fusion of a camelid-derived V H H to a bovine Fc where x is either 1, 3, 9, or 10, corresponding to the isolated V H Hs; SC, bovine secretory component; JC, bovine JC; c-Myc, FLAG, HA, detection tags; KDEL, endoplasmic reticulum retrieval tetra-peptide; CPMV 3′UTR, 3′-untranslated region of Cowpea mosaic virus; nos, nopaline synthase terminator sequence; the cassettes were cloned into pEAQ-DEST-1 plant expression vectors. Schematic not drawn to scale. Bold outlines indicate translated regions. (C) Monovalent affinities and kinetics of the interaction between V H Hs and MBP-Int277 by SPR (pH 7.4, 25°C). (D) Predicted protein size and number of glycosylation sites for each subunit. (E–G) Western blots of crude extract from leaves of N. benthamiana harvested at 6 dpi expressing V H H1, 3, 9, and 10-Fc along with p19, a suppressor of gene silencing (E) , SC (F) , and JC (G) . 10 μg of TSP was loaded in each lane.

    Journal: Frontiers in Plant Science

    Article Title: Plant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells

    doi: 10.3389/fpls.2019.00270

    Figure Lengend Snippet: Design and production of individual subunits required for chimeric secretory IgA assembly. (A) Schematic of all produced subunits fully assembled into a chimeric antibody intended for secretory IgA functionality. It notably differs from the structure of native secretory IgA by the replacement of the Fab region with a camelid-derived variable heavy chain fragment (V H H). (B) Schematic representation of constructs used for Agrobacterium -mediated transient expression in N. benthamiana leaves. CaMV 35S, cauliflower mosaic virus 35S promoter; CPMV 5′UTR, 5′-untranslated region of Cowpea mosaic virus; PR1b, tobacco pathogenesis-related protein 1b signal peptide; V H Hx-Fc, fusion of a camelid-derived V H H to a bovine Fc where x is either 1, 3, 9, or 10, corresponding to the isolated V H Hs; SC, bovine secretory component; JC, bovine JC; c-Myc, FLAG, HA, detection tags; KDEL, endoplasmic reticulum retrieval tetra-peptide; CPMV 3′UTR, 3′-untranslated region of Cowpea mosaic virus; nos, nopaline synthase terminator sequence; the cassettes were cloned into pEAQ-DEST-1 plant expression vectors. Schematic not drawn to scale. Bold outlines indicate translated regions. (C) Monovalent affinities and kinetics of the interaction between V H Hs and MBP-Int277 by SPR (pH 7.4, 25°C). (D) Predicted protein size and number of glycosylation sites for each subunit. (E–G) Western blots of crude extract from leaves of N. benthamiana harvested at 6 dpi expressing V H H1, 3, 9, and 10-Fc along with p19, a suppressor of gene silencing (E) , SC (F) , and JC (G) . 10 μg of TSP was loaded in each lane.

    Article Snippet: The recombinant proteins were detected with one of the following primary antibodies: mouse anti-c-Myc monoclonal antibody (GenScript, Cat. No. A00864), mouse anti-HA monoclonal antibody (Millipore Sigma, Cat. No. H3663), mouse anti-FLAG monoclonal antibody (Millipore Sigma, Cat. No. F3165), and HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad, Cat. No. 170-6516).

    Techniques: Produced, Derivative Assay, Construct, Expressing, Isolation, Sequencing, Clone Assay, SPR Assay, Western Blot

    Relative stability of NCPs containing H3 and H3.3. ( A ) NCPs were prepared from cells expressing either H3-Flag or H3.3-Flag. NCP monomers suspended in a solvent containing 150 mM NaCl were obtained by fractionation on a sucrose gradient (see Materials and Methods) in a solvent containing 80 mM NaCl and buffers. In each case, the sample was immunoprecipitated with antibody to Flag, histones were isolated and fractionated by gel electrophoresis, and Western blots of the samples were probed with antibody to H2A ( left ) or H2A.Z ( right ). Input lanes are loaded with an aliquot representing 10% of the starting sample. The label “150 mM/80 mM gradient” indicates the highest NaCl concentration used in NCP preparation and the NaCl concentration in the gradient. (Nab) No antibody control. ( B ) Comparative recoveries of H2A and H2A.Z from the data in A . The amounts of H2A or H2A.Z in H3-Flag-containing NCPs were set to 1. The relative amount was calculated by comparing the intensity of immunoprecipitated H2A or H2A.Z with that of immunoprecipitated H3-Flag or H3.3-Flag.

    Journal: Genes & Development

    Article Title: Nucleosome stability mediated by histone variants H3.3 and H2A.Z

    doi: 10.1101/gad.1547707

    Figure Lengend Snippet: Relative stability of NCPs containing H3 and H3.3. ( A ) NCPs were prepared from cells expressing either H3-Flag or H3.3-Flag. NCP monomers suspended in a solvent containing 150 mM NaCl were obtained by fractionation on a sucrose gradient (see Materials and Methods) in a solvent containing 80 mM NaCl and buffers. In each case, the sample was immunoprecipitated with antibody to Flag, histones were isolated and fractionated by gel electrophoresis, and Western blots of the samples were probed with antibody to H2A ( left ) or H2A.Z ( right ). Input lanes are loaded with an aliquot representing 10% of the starting sample. The label “150 mM/80 mM gradient” indicates the highest NaCl concentration used in NCP preparation and the NaCl concentration in the gradient. (Nab) No antibody control. ( B ) Comparative recoveries of H2A and H2A.Z from the data in A . The amounts of H2A or H2A.Z in H3-Flag-containing NCPs were set to 1. The relative amount was calculated by comparing the intensity of immunoprecipitated H2A or H2A.Z with that of immunoprecipitated H3-Flag or H3.3-Flag.

    Article Snippet: The antibodies used were as follows: anti-Flag M2 Monoclonal antibody (F3165), anti-histone H2A.Z (07-594), anti-histone H3 (05-499), anti-acetyl-histone H3 (06-599), anti-histone H2A (7-146), and anti-histone H2B (07-371).

    Techniques: Expressing, Fractionation, Immunoprecipitation, Isolation, Nucleic Acid Electrophoresis, Western Blot, Concentration Assay

    ( A ) ChIP analysis of H3.3-Flag and H2A.Z over distal promoter or enhancer regions and transcribed regions of a variety of genes in 6C2 cells expressing H3.3-Flag. (Open bars) No antibody control; (filled bars) anti-Flag or anti-H2A.Z immunoprecipitation. Error bars reflect three separate measurements. (PAI) Plasminogen activator inhibitor; (FOG) friend of GATA. ( B ) Double ChIP analysis over same regions. First ChIPs by anti-Flag were followed by second ChIPs by anti-H2A.Z antibodies. ( C ) Summary of ChIP and double ChIP results; level of Ac/H3K9 K14; relative expression level of those genes surveyed in wild-type 6C2 cells and in the cells overexpressing untagged H3.3. The ChIP data are from A and B ). (N/A) Not applicable. ( D ) Schematic representation of relative stability of nucleosomes containing different histone variants.

    Journal: Genes & Development

    Article Title: Nucleosome stability mediated by histone variants H3.3 and H2A.Z

    doi: 10.1101/gad.1547707

    Figure Lengend Snippet: ( A ) ChIP analysis of H3.3-Flag and H2A.Z over distal promoter or enhancer regions and transcribed regions of a variety of genes in 6C2 cells expressing H3.3-Flag. (Open bars) No antibody control; (filled bars) anti-Flag or anti-H2A.Z immunoprecipitation. Error bars reflect three separate measurements. (PAI) Plasminogen activator inhibitor; (FOG) friend of GATA. ( B ) Double ChIP analysis over same regions. First ChIPs by anti-Flag were followed by second ChIPs by anti-H2A.Z antibodies. ( C ) Summary of ChIP and double ChIP results; level of Ac/H3K9 K14; relative expression level of those genes surveyed in wild-type 6C2 cells and in the cells overexpressing untagged H3.3. The ChIP data are from A and B ). (N/A) Not applicable. ( D ) Schematic representation of relative stability of nucleosomes containing different histone variants.

    Article Snippet: The antibodies used were as follows: anti-Flag M2 Monoclonal antibody (F3165), anti-histone H2A.Z (07-594), anti-histone H3 (05-499), anti-acetyl-histone H3 (06-599), anti-histone H2A (7-146), and anti-histone H2B (07-371).

    Techniques: Chromatin Immunoprecipitation, Expressing, Immunoprecipitation

    Progranulin Interacts with GCase. HEK-293 cells were co-transfected with constructs expressing HA-tagged human progranulin and/or myc-flag-tagged human GCase. HA-tagged progranulin was then immunoprecipitated from cell lysates with an anti-HA antibody. a , Flag-tagged GCase co-immunoprecipitated with progranulin, indicating interaction of the two proteins. b , Consistent with the co-immunoprecipitation of GCase with progranulin, we detected strong proximity ligation (PLA) signal in HEK-293 cells co-transfected with human progranulin and human GCase constructs. c , The specificity of the Flag-HA PLA signal was confirmed by the presence of significantly more PLA puncta from cells co-transfected with the progranulin-HA and GCase-Flag constructs than in cells transfected with only one of the constructs, or from cells that underwent PLA in the absence of the HA and Flag antibodies (ANOVA effect of experimental condition, p

    Journal: Acta Neuropathologica Communications

    Article Title: Impaired β-glucocerebrosidase activity and processing in frontotemporal dementia due to progranulin mutations

    doi: 10.1186/s40478-019-0872-6

    Figure Lengend Snippet: Progranulin Interacts with GCase. HEK-293 cells were co-transfected with constructs expressing HA-tagged human progranulin and/or myc-flag-tagged human GCase. HA-tagged progranulin was then immunoprecipitated from cell lysates with an anti-HA antibody. a , Flag-tagged GCase co-immunoprecipitated with progranulin, indicating interaction of the two proteins. b , Consistent with the co-immunoprecipitation of GCase with progranulin, we detected strong proximity ligation (PLA) signal in HEK-293 cells co-transfected with human progranulin and human GCase constructs. c , The specificity of the Flag-HA PLA signal was confirmed by the presence of significantly more PLA puncta from cells co-transfected with the progranulin-HA and GCase-Flag constructs than in cells transfected with only one of the constructs, or from cells that underwent PLA in the absence of the HA and Flag antibodies (ANOVA effect of experimental condition, p

    Article Snippet: Immunoprecipitates were blotted for Flag tag (mouse monoclonal, #F3165, MilliporeSigma) to detect co-immunoprecipitated GCase, and for progranulin to confirm successful pull down of HA-tagged progranulin (rabbit polyclonal, #40–3400, ThermoFisher).

    Techniques: Transfection, Construct, Expressing, Immunoprecipitation, Ligation, Proximity Ligation Assay

    Smad4 provides an example of a protein that must be methylated before it can be phosphorylated by GSK3 and translocated into MVBs by Wnt signaling. ( A ) Diagram of how FGF/EGF, Wnt, and TGF-β/BMP signaling cross-talk at the level of Smad4. MAPK/FGF (green) primes phosphorylation by GSK3 (blue) at three sites; the meArg site discovered in this study is shown in red. ( B ) Wnt addition for 20 min increased Smad4 methylation in transfected HEK-293T cells. S4-Flag and GAPDH serve as loading controls. ( C – E ) Phospho-Smad4-Flag relocalized to vesicular structures after 15 min of Wnt3a addition, but only in the absence of the competitive methylation inhibitor Adox. ( F ) A potential Smad4 arginine-methylation site was mutated (R272K) to prevent arginine methylation with minimal effect on the protein. ( G ) Smad4-Flag-WT immunoprecipitated from transfected HEK-293T lysates was recognized by asymmetric dimethyl-Arg antibody while the Smad4-Flag-R272K mutant was not. Thus, Smad4 contains a single meArg site. ( H ) Smad4 phosphorylation by GSK3 requires arginine methylation. Ratios under each lane and the merge panels indicate GSK3 phosphorylated Smad4/total Smad4-Flag. ( I – N ) In situ protease protection assay using digitonin and proteinase K showing that wild-type Smad4-Flag was translocated inside membrane-bound organelles when Wnt was added for 15 min ( J ) but digested when Triton X-100 was added ( K ). Smad4-R272K-Flag was not translocated into membrane vesicles and was degraded by proteinase K ( L and M ). Panels I′ – N′ show DAPI staining and differential interference contrast microscopy to visualize cellular contours in the corresponding cells shown above. ( O ) Smad4 wild type (WT), Smad4-R272K, and Smad4 mutated at the three GSK3 sites (phosphorylation-resistant Smad4-GM) were tested in TGF-β signaling assays. HaCaT cells permanently transfected with the CAGA12-luciferase reporter and constitutive CMV-Renilla (in which MAPK activation was primed by addition of EGF) were used. This indicates that in the context of TGF-β signaling arginine methylation is required for the integration of FGF, Wnt, and TGF-β signals. ** P

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

    Article Title: Arginine methylation is required for canonical Wnt signaling and endolysosomal trafficking

    doi: 10.1073/pnas.1804091115

    Figure Lengend Snippet: Smad4 provides an example of a protein that must be methylated before it can be phosphorylated by GSK3 and translocated into MVBs by Wnt signaling. ( A ) Diagram of how FGF/EGF, Wnt, and TGF-β/BMP signaling cross-talk at the level of Smad4. MAPK/FGF (green) primes phosphorylation by GSK3 (blue) at three sites; the meArg site discovered in this study is shown in red. ( B ) Wnt addition for 20 min increased Smad4 methylation in transfected HEK-293T cells. S4-Flag and GAPDH serve as loading controls. ( C – E ) Phospho-Smad4-Flag relocalized to vesicular structures after 15 min of Wnt3a addition, but only in the absence of the competitive methylation inhibitor Adox. ( F ) A potential Smad4 arginine-methylation site was mutated (R272K) to prevent arginine methylation with minimal effect on the protein. ( G ) Smad4-Flag-WT immunoprecipitated from transfected HEK-293T lysates was recognized by asymmetric dimethyl-Arg antibody while the Smad4-Flag-R272K mutant was not. Thus, Smad4 contains a single meArg site. ( H ) Smad4 phosphorylation by GSK3 requires arginine methylation. Ratios under each lane and the merge panels indicate GSK3 phosphorylated Smad4/total Smad4-Flag. ( I – N ) In situ protease protection assay using digitonin and proteinase K showing that wild-type Smad4-Flag was translocated inside membrane-bound organelles when Wnt was added for 15 min ( J ) but digested when Triton X-100 was added ( K ). Smad4-R272K-Flag was not translocated into membrane vesicles and was degraded by proteinase K ( L and M ). Panels I′ – N′ show DAPI staining and differential interference contrast microscopy to visualize cellular contours in the corresponding cells shown above. ( O ) Smad4 wild type (WT), Smad4-R272K, and Smad4 mutated at the three GSK3 sites (phosphorylation-resistant Smad4-GM) were tested in TGF-β signaling assays. HaCaT cells permanently transfected with the CAGA12-luciferase reporter and constitutive CMV-Renilla (in which MAPK activation was primed by addition of EGF) were used. This indicates that in the context of TGF-β signaling arginine methylation is required for the integration of FGF, Wnt, and TGF-β signals. ** P

    Article Snippet: Antibodies were obtained from the following sources: mouse monoclonal antibody against PRMT1 (Santa Cruz Biotechnology, sc-59648; 1:1,000 for immunostaining and Western blots); antibody against Lamp1 (Cell Signaling, 3243; 1:1,000 for immunostaining); antibodies against asymmetric dimethylarginine modifications (MilliporeSigma, ASYM24; 1:500 for immunostaining or Western blots); and (Abcam 21C7; 1:500 for immunostaining or Western blots); antibody against GSK3 (BD Biosciences, 610201; 1:500 for immunostaining and 1:1,000 for Western blots); antibody against K48-polyubiquitin (MilliporeSigma, 05–1307; 1:1,000 for immunostaining); antibody against Flag (MilliporeSigma, F1804; 1:5,000 for immunostaining or Western blots); antibody against GAPDH (Cell Signaling Technology, 2118; 1:1,000 for Western blots); and actin (MilliporeSigma, A2066; 1:1,000 for Western blots) were used as loading controls for Western blots.

    Techniques: Methylation, Transfection, Immunoprecipitation, Mutagenesis, In Situ, Staining, Microscopy, Luciferase, Activation Assay