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Bethyl brap2

Figure 1. <t>Brap2</t> associates with Nedd8. (A) Schematic structure of the Nedd8 constructs for yeast two-hybrid screening. (B) Yeast PJ69-4A strains were transformed with expression vectors as indicated. Individual transformants were streaked to synthetic medium plates lacking tryptophan, leucine, histidine. (C) HEK293 cells were transfected with expression vectors as indicated and were subjected to immuno- precipitation (IP) with indicated antibodies. The total cell lysates and immunoprecipitants (IP) were subjected to immunoblot (IB) analyses with antibodies to Flag and HA. doi:10.1371/journal.pone.0058911.g001

Brap2, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response."

Article Title: Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response.

Journal: PloS one

doi: 10.1371/journal.pone.0058911

Figure 1. Brap2 associates with Nedd8. (A) Schematic structure of the Nedd8 constructs for yeast two-hybrid screening. (B) Yeast PJ69-4A strains were transformed with expression vectors as indicated. Individual transformants were streaked to synthetic medium plates lacking tryptophan, leucine, histidine. (C) HEK293 cells were transfected with expression vectors as indicated and were subjected to immuno- precipitation (IP) with indicated antibodies. The total cell lysates and immunoprecipitants (IP) were subjected to immunoblot (IB) analyses with antibodies to Flag and HA. doi:10.1371/journal.pone.0058911.g001

Figure Legend Snippet: Figure 1. Brap2 associates with Nedd8. (A) Schematic structure of the Nedd8 constructs for yeast two-hybrid screening. (B) Yeast PJ69-4A strains were transformed with expression vectors as indicated. Individual transformants were streaked to synthetic medium plates lacking tryptophan, leucine, histidine. (C) HEK293 cells were transfected with expression vectors as indicated and were subjected to immuno- precipitation (IP) with indicated antibodies. The total cell lysates and immunoprecipitants (IP) were subjected to immunoblot (IB) analyses with antibodies to Flag and HA. doi:10.1371/journal.pone.0058911.g001

Techniques Used: Construct, Two Hybrid Screening, Transformation Assay, Expressing, Transfection, Immunoprecipitation, Western Blot

Figure 2. The RING domain of Brap2 is responsible for the interaction with Nedd8. (A) Schematic structure of deletion mutants of Brap2. (B) HEK293 cells were transfected with indicated plasmids and were subjected to immunoprecipitation (IP). The immunoprecipitants (IP) and the total cell lysates were immunoblotted (IB) with indicated antibodies. (C) Schematic structure of point mutant of Brap2. (D) HEK293 cells were transfected with expression vectors as indicated and were subjected to IP. (E) HEK293 cells were transfected with HA-Nedd8 and Flag-Brap2, immunoprecipitated with Flag antibody. The total cell lysates and IP were immunoblotted with HA antibody. doi:10.1371/journal.pone.0058911.g002

Figure Legend Snippet: Figure 2. The RING domain of Brap2 is responsible for the interaction with Nedd8. (A) Schematic structure of deletion mutants of Brap2. (B) HEK293 cells were transfected with indicated plasmids and were subjected to immunoprecipitation (IP). The immunoprecipitants (IP) and the total cell lysates were immunoblotted (IB) with indicated antibodies. (C) Schematic structure of point mutant of Brap2. (D) HEK293 cells were transfected with expression vectors as indicated and were subjected to IP. (E) HEK293 cells were transfected with HA-Nedd8 and Flag-Brap2, immunoprecipitated with Flag antibody. The total cell lysates and IP were immunoblotted with HA antibody. doi:10.1371/journal.pone.0058911.g002

Techniques Used: Transfection, Immunoprecipitation, Mutagenesis, Expressing

Figure 3. Brap2 is neddylated at lysine-432 in vivo. (A) Alignment of putative neddylation site of Brap2 with the consensus neddylation site of cullin family proteins. (B) HEK293 cells were transfected with indicated plasmids and were subjected to immunoprecipitation (IP) with Flag antibody and the resulting immunoprecipitant (IP) and total cell lysates were immnoblotted (IB) with indicated antibodies. doi:10.1371/journal.pone.0058911.g003

Figure Legend Snippet: Figure 3. Brap2 is neddylated at lysine-432 in vivo. (A) Alignment of putative neddylation site of Brap2 with the consensus neddylation site of cullin family proteins. (B) HEK293 cells were transfected with indicated plasmids and were subjected to immunoprecipitation (IP) with Flag antibody and the resulting immunoprecipitant (IP) and total cell lysates were immnoblotted (IB) with indicated antibodies. doi:10.1371/journal.pone.0058911.g003

Techniques Used: In Vivo, Transfection, Immunoprecipitation

Figure 4. Brap2 dimerizes independent of Nedd8. (A) HEK293 cells were transfected with indicated plasmids and were then subjected to immunoprecipitation with Flag antibody. The total cell lysate and immunoprecipitant (IP) were immunoblotted (IB) using HA and Flag antibodies. Asterisks indicate non-specific bands. (B) HEK293 cells were transfected with indicated plasmids and incubated for 6 hours in the presence or absence of 5 mg/ml MLN4924, and were then subjected immunoprecipitation with HA antibody. The total cell lysate and immunoprecipitant (IP) were immunoblotted (IB) using HA and Flag antibodies. Asterisk indicates non-specific bands. doi:10.1371/journal.pone.0058911.g004

Figure Legend Snippet: Figure 4. Brap2 dimerizes independent of Nedd8. (A) HEK293 cells were transfected with indicated plasmids and were then subjected to immunoprecipitation with Flag antibody. The total cell lysate and immunoprecipitant (IP) were immunoblotted (IB) using HA and Flag antibodies. Asterisks indicate non-specific bands. (B) HEK293 cells were transfected with indicated plasmids and incubated for 6 hours in the presence or absence of 5 mg/ml MLN4924, and were then subjected immunoprecipitation with HA antibody. The total cell lysate and immunoprecipitant (IP) were immunoblotted (IB) using HA and Flag antibodies. Asterisk indicates non-specific bands. doi:10.1371/journal.pone.0058911.g004

Techniques Used: Transfection, Immunoprecipitation, Incubation

Figure 5. Brap2 associates with Cul1 in a neddylation-independent manner. (A) HEK293 cells were transfected with HA-Cul1 and Flag-Brap2 WT or CA. HA-Cul1 was immunoprecipitated and total cell lysates and immunoprecipitants (IP) were immunoblotted (IB) with Flag and HA antibodies. (B) HEK293T cells were transfected with indicated plasmids and incubated for 1 hour in the presence or absence of 20 mM MG132, and subjected to immunoprecipitation (IP) analysis. Asterisk indicates non-specific bands. (C) HEK293T cells were transfected with indicated plasmids and incubated for 4 hours in the presence or absence of 1 mM MLN4924, and subjected to immunoprecipitation (IP). The immunoprecipitants (IP) and the total cell lysates were immunoblotted (IB) with indicated antibodies. doi:10.1371/journal.pone.0058911.g005

Figure Legend Snippet: Figure 5. Brap2 associates with Cul1 in a neddylation-independent manner. (A) HEK293 cells were transfected with HA-Cul1 and Flag-Brap2 WT or CA. HA-Cul1 was immunoprecipitated and total cell lysates and immunoprecipitants (IP) were immunoblotted (IB) with Flag and HA antibodies. (B) HEK293T cells were transfected with indicated plasmids and incubated for 1 hour in the presence or absence of 20 mM MG132, and subjected to immunoprecipitation (IP) analysis. Asterisk indicates non-specific bands. (C) HEK293T cells were transfected with indicated plasmids and incubated for 4 hours in the presence or absence of 1 mM MLN4924, and subjected to immunoprecipitation (IP). The immunoprecipitants (IP) and the total cell lysates were immunoblotted (IB) with indicated antibodies. doi:10.1371/journal.pone.0058911.g005

Techniques Used: Transfection, Immunoprecipitation, Incubation

Figure 6. Brap2 attenuates TNF-a-induced NF-kB translocation to nucleus. (A) HeLa cells were transfected with either Flag-Brap2 WT or Flag-Brap2 CA and stimulated with or without 5 ng/ml TNF-a for 30 min, and were subjected to immunocytochemistry using anti-Flag or anti-RelA/ p65 antibodies. Cells that express certain amount of Flag-Brap2 are marked by dot lines and control cells are marked by solid line. RelA/p65 translocates in nucleus after the stimulation. The right panels show the same cells using a rainbow color. Bars, 50 mm. (B) Ratiometric measurement of RelA/p65 fluorescence observed in cells expressing Flag-Brap2 before and after treatment with 5 ng/ml TNF-a (n = 50; mean 6 SEM; *** p,0.0001 by one-way ANOVA). (C) HEK293T cells were transfected with HA-Cul1 and Flag-Brap2 WT or CA, and stimulated with or without 5 ng/ml TNF-a for the indicated time, and subjected to immunoprecipitation (IP) with Flag antibody. The total cell lysates and IP were immunoblotted (IB) with indicated antibodies. (D) HEK293 cells were stimulated with 5 ng/ml TNF-a for indicated time, and cell lysates were subjected to immunoblot analysis. doi:10.1371/journal.pone.0058911.g006

Figure Legend Snippet: Figure 6. Brap2 attenuates TNF-a-induced NF-kB translocation to nucleus. (A) HeLa cells were transfected with either Flag-Brap2 WT or Flag-Brap2 CA and stimulated with or without 5 ng/ml TNF-a for 30 min, and were subjected to immunocytochemistry using anti-Flag or anti-RelA/ p65 antibodies. Cells that express certain amount of Flag-Brap2 are marked by dot lines and control cells are marked by solid line. RelA/p65 translocates in nucleus after the stimulation. The right panels show the same cells using a rainbow color. Bars, 50 mm. (B) Ratiometric measurement of RelA/p65 fluorescence observed in cells expressing Flag-Brap2 before and after treatment with 5 ng/ml TNF-a (n = 50; mean 6 SEM; *** p,0.0001 by one-way ANOVA). (C) HEK293T cells were transfected with HA-Cul1 and Flag-Brap2 WT or CA, and stimulated with or without 5 ng/ml TNF-a for the indicated time, and subjected to immunoprecipitation (IP) with Flag antibody. The total cell lysates and IP were immunoblotted (IB) with indicated antibodies. (D) HEK293 cells were stimulated with 5 ng/ml TNF-a for indicated time, and cell lysates were subjected to immunoblot analysis. doi:10.1371/journal.pone.0058911.g006

Techniques Used: Translocation Assay, Transfection, Immunocytochemistry, Control, Fluorescence, Expressing, Immunoprecipitation, Western Blot

Figure 7. Neddylation of Brap2 is associated with TNF-a-induced NF-kB activity. (A) TNF-a-dependent activation of a NF-kB reporter gene in HEK293 cells expressing indicated plasmids. Cells were stimulated with 5 ng/ml TNF-a for 3 hours. (n = 3; mean 6 SEM; *** p,0.0001 by one-way ANOVA). (B) TNF-a-dependent activation of a NF-kB reporter gene in HEK293 cells expressing indicated plasmids. Cells were stimulated with 5 ng/ml TNF-a for 6 hours. (n = 3; mean 6 SEM; n.s., not significant by one-way ANOVA). doi:10.1371/journal.pone.0058911.g007

Figure Legend Snippet: Figure 7. Neddylation of Brap2 is associated with TNF-a-induced NF-kB activity. (A) TNF-a-dependent activation of a NF-kB reporter gene in HEK293 cells expressing indicated plasmids. Cells were stimulated with 5 ng/ml TNF-a for 3 hours. (n = 3; mean 6 SEM; *** p,0.0001 by one-way ANOVA). (B) TNF-a-dependent activation of a NF-kB reporter gene in HEK293 cells expressing indicated plasmids. Cells were stimulated with 5 ng/ml TNF-a for 6 hours. (n = 3; mean 6 SEM; n.s., not significant by one-way ANOVA). doi:10.1371/journal.pone.0058911.g007

Techniques Used: Activity Assay, Activation Assay, Expressing

Figure 8. Model of Brap2 function. (A) Model of Brap2 interaction with Cul1 and its effect on NF-kB signaling pathway. (B) Time course of NF-kB translocation and target gene expression by each Brap2 constructs. doi:10.1371/journal.pone.0058911.g008

Figure Legend Snippet: Figure 8. Model of Brap2 function. (A) Model of Brap2 interaction with Cul1 and its effect on NF-kB signaling pathway. (B) Time course of NF-kB translocation and target gene expression by each Brap2 constructs. doi:10.1371/journal.pone.0058911.g008

Techniques Used: Translocation Assay, Targeted Gene Expression, Construct

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(a) Western analysis for expression of BRAP2 protein in normal mouse tissues and in HeLa/GC2 cell lines performed as per the methods section. (b) Classification of potential BRAP2 binding proteins identified in Y2H screen according to broad functions as per data mining.

Journal: Scientific Reports

Article Title: Interactome of the negative regulator of nuclear import BRCA1-binding protein 2

doi: 10.1038/srep09459

Figure Lengend Snippet: (a) Western analysis for expression of BRAP2 protein in normal mouse tissues and in HeLa/GC2 cell lines performed as per the methods section. (b) Classification of potential BRAP2 binding proteins identified in Y2H screen according to broad functions as per data mining.

Article Snippet: Co-immunoprecipitation was performed from 400 μg of mouse testis lysate using the Catch and Release® v2.0 Reversible Immunoprecipitation System (Upstate Cell Signalling Solutions) according to the manufacturer's instructions, using 4 μg of anti-PHLPP1 (Novus Biologicals), anti-AKAP3 (Protein tech), anti-DNMT1 (Abcam), anti-BRAP2 (Sigma), or anti-GST (Santa Cruz) antibodies.

Techniques: Western Blot, Expressing, Binding Assay

List of BRAP2 interacting proteins identified in this study. A Y2H screen was performed on a human testis cDNA library using human BRAP2 (343–592) as a bait to identify BRAP2 interactors. Predicted biological score (PBS) indicates confidence of interaction (A is highest); selective interactive domain (SID) is the minimal sequence shared by all fragments for the binding partner identified that interact with BRAP2

Journal: Scientific Reports

Article Title: Interactome of the negative regulator of nuclear import BRCA1-binding protein 2

doi: 10.1038/srep09459

Figure Lengend Snippet: List of BRAP2 interacting proteins identified in this study. A Y2H screen was performed on a human testis cDNA library using human BRAP2 (343–592) as a bait to identify BRAP2 interactors. Predicted biological score (PBS) indicates confidence of interaction (A is highest); selective interactive domain (SID) is the minimal sequence shared by all fragments for the binding partner identified that interact with BRAP2

Techniques: cDNA Library Assay, Sequencing, Binding Assay, Modification, Activity Assay, RNA Binding Assay

$(a) Coimmunoprecipitation (IP) experiments were performed as described in methods from adult mouse testis lysate using anti-PHLPP1, AKAP3, DNMT1 or GST antibodies and subjected to Western analysis using anti-BRAP2 (left) where as anti-BRAP2 antibody was used for IP from the same lysates and subjected to Western analysis using anti-PHLPP1, AKAP3 or anti-GST antibody (right) (b) GFP pull downs were performed from Hek293T cells co-transfected to express either GFP-BRAP2 (343-592), or GFP and HA-PHLPP1 α, or HA-PHLPP1 β or GST-AKAP3 or Myc-DNMT1 prior to Western analysis of GFP-trap-precipitated (bound) fractions using specific antibodies to GFP.$

Journal: Scientific Reports

Article Title: Interactome of the negative regulator of nuclear import BRCA1-binding protein 2

doi: 10.1038/srep09459

Figure Lengend Snippet: (a) Coimmunoprecipitation (IP) experiments were performed as described in methods from adult mouse testis lysate using anti-PHLPP1, AKAP3, DNMT1 or GST antibodies and subjected to Western analysis using anti-BRAP2 (left) where as anti-BRAP2 antibody was used for IP from the same lysates and subjected to Western analysis using anti-PHLPP1, AKAP3 or anti-GST antibody (right) (b) GFP pull downs were performed from Hek293T cells co-transfected to express either GFP-BRAP2 (343-592), or GFP and HA-PHLPP1 α, or HA-PHLPP1 β or GST-AKAP3 or Myc-DNMT1 prior to Western analysis of GFP-trap-precipitated (bound) fractions using specific antibodies to GFP.

Techniques: Western Blot, Transfection

(a) Schematic representation of domain structure of BRAP2 and DsRed2 fused BRAP2 343–592 and 442–592. Amino acid residue numbers are indicated (top) Ring domain = 263–303; Zinc-finger ubiquitin binding domain (ZnF UBP) = 315–364; Coiled coil domain = 430–535. (b) CLSM images of HeLa cells transfected to express DsRed2 vector or DsRed2 fused BRAP2 constructs (as indicated) followed by fixation 20 hours post transfection and immunostained for endogenous PHLPP1 (left panels). Quantitative analysis on digitized CLSM images (right panel) to determine the Fn/c ratio (nuclear to cytoplasmic fluorescence ratio, above background) for endogenous PHLPP1 protein in presence and absence of indicated DsRed2 fused proteins. Values represent the mean +/− SEM (n > 30), with p values (Student's t test) shown where there were significant differences between values in absence or presence of the BRAP2 constructs (right). (ci) As per (b), CLSM images of HeLa cells immunostained with DNMT1 antibody in presence or absence of ectopically expressed DsRed2 fused BRAP2 constructs (as indicated on panels) or DsRed2 vector (left) together with quantitative analysis (right panel). (cii) As per (b), CLSM images of live HeLa cells co-transfected for ectopic expression of GFP-DNMT1 with DsRed2 or DsRed2 fused BRAP2 343–592 and 442–570 post 20–24 hours (left), together with quantitative analysis (right panel). (d) As per (b), CLSM images of Cos7 cells immunostained with T-ag antibody in presence or absence of ectopically expressed DsRed2 fused BRAP2 constructs (as indicated on panels) or DsRed2 vector (left) together with quantitative analysis (right panel).

Journal: Scientific Reports

Article Title: Interactome of the negative regulator of nuclear import BRCA1-binding protein 2

doi: 10.1038/srep09459

Figure Lengend Snippet: (a) Schematic representation of domain structure of BRAP2 and DsRed2 fused BRAP2 343–592 and 442–592. Amino acid residue numbers are indicated (top) Ring domain = 263–303; Zinc-finger ubiquitin binding domain (ZnF UBP) = 315–364; Coiled coil domain = 430–535. (b) CLSM images of HeLa cells transfected to express DsRed2 vector or DsRed2 fused BRAP2 constructs (as indicated) followed by fixation 20 hours post transfection and immunostained for endogenous PHLPP1 (left panels). Quantitative analysis on digitized CLSM images (right panel) to determine the Fn/c ratio (nuclear to cytoplasmic fluorescence ratio, above background) for endogenous PHLPP1 protein in presence and absence of indicated DsRed2 fused proteins. Values represent the mean +/− SEM (n > 30), with p values (Student's t test) shown where there were significant differences between values in absence or presence of the BRAP2 constructs (right). (ci) As per (b), CLSM images of HeLa cells immunostained with DNMT1 antibody in presence or absence of ectopically expressed DsRed2 fused BRAP2 constructs (as indicated on panels) or DsRed2 vector (left) together with quantitative analysis (right panel). (cii) As per (b), CLSM images of live HeLa cells co-transfected for ectopic expression of GFP-DNMT1 with DsRed2 or DsRed2 fused BRAP2 343–592 and 442–570 post 20–24 hours (left), together with quantitative analysis (right panel). (d) As per (b), CLSM images of Cos7 cells immunostained with T-ag antibody in presence or absence of ectopically expressed DsRed2 fused BRAP2 constructs (as indicated on panels) or DsRed2 vector (left) together with quantitative analysis (right panel).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Construct, Fluorescence, Expressing

Immunohistochemistry of Bouins fixed paraffin embedded adult mouse testis sections from Asmu:Swiss mouse was performed for BRAP2, PHLPP1, AKAP3 and DNMT1. BRAP2 expression was observed in the cytoplasm of pachytene spermatocytes (PS), round (RS) and elongated spermatids (ES) as indicated (arrows). PHLPP1 was observed in nuclei of spermatogonia and is cytoplasmic in pachytene spermatocytes and round spermatids. AKAP3 was present in the cytoplasm of round and elongated spermatids. DNMT1 was present in the nuclei of spermatogonia and both in nucleus and cytoplasm of pachytene spermatocytes and only in cytoplasm of round and elongated spermatids. The panels at the right represents respective negative control showing testis sections with no primary antibody .

Journal: Scientific Reports

Article Title: Interactome of the negative regulator of nuclear import BRCA1-binding protein 2

doi: 10.1038/srep09459

Figure Lengend Snippet: Immunohistochemistry of Bouins fixed paraffin embedded adult mouse testis sections from Asmu:Swiss mouse was performed for BRAP2, PHLPP1, AKAP3 and DNMT1. BRAP2 expression was observed in the cytoplasm of pachytene spermatocytes (PS), round (RS) and elongated spermatids (ES) as indicated (arrows). PHLPP1 was observed in nuclei of spermatogonia and is cytoplasmic in pachytene spermatocytes and round spermatids. AKAP3 was present in the cytoplasm of round and elongated spermatids. DNMT1 was present in the nuclei of spermatogonia and both in nucleus and cytoplasm of pachytene spermatocytes and only in cytoplasm of round and elongated spermatids. The panels at the right represents respective negative control showing testis sections with no primary antibody .

Techniques: Immunohistochemistry, Expressing, Negative Control

The localisation of BRAP2 and AKAP3 was examined in sperm collected from the epididymis of C57 Black adult wild type mice by indirect immunofluorescence microscopy using antibodies specific for BRAP2 and AKAP3 as per methods. Micrographs were taken ×100 magnification. h, sperm head, mp, mid piece; pp, principal piece; av, acrosomal vesicle; pa, post acrosomal region.

Journal: Scientific Reports

Article Title: Interactome of the negative regulator of nuclear import BRCA1-binding protein 2

doi: 10.1038/srep09459

Figure Lengend Snippet: The localisation of BRAP2 and AKAP3 was examined in sperm collected from the epididymis of C57 Black adult wild type mice by indirect immunofluorescence microscopy using antibodies specific for BRAP2 and AKAP3 as per methods. Micrographs were taken ×100 magnification. h, sperm head, mp, mid piece; pp, principal piece; av, acrosomal vesicle; pa, post acrosomal region.

Techniques: Immunofluorescence, Microscopy

In cell types such as the early spermatogonia where BRAP2 (blue) is expressed at low levels, BRAP2 binding partners such as PHLPP1 (red) and DNMT1 (green) are nuclear. Higher BRAP2 expression in the later germ cell types (pachytene spermatocytes, round and elongated spermatids), results in inhibition of nuclear targeting of PHLPP1 and DNMT1. BRAP2 protein persists late into spermiogenesis, being still present in the mature spermatozoan and hence able to interact with binding partners such as AKAP3 (yellow), which reach maximum levels in the mature spermatozoan. BRAP2 would appear to play a more structural/scaffold role to help localise proteins such as AKAP3 in the post acrosomal space and principal piece of sperm.

Journal: Scientific Reports

Article Title: Interactome of the negative regulator of nuclear import BRCA1-binding protein 2

doi: 10.1038/srep09459

Figure Lengend Snippet: In cell types such as the early spermatogonia where BRAP2 (blue) is expressed at low levels, BRAP2 binding partners such as PHLPP1 (red) and DNMT1 (green) are nuclear. Higher BRAP2 expression in the later germ cell types (pachytene spermatocytes, round and elongated spermatids), results in inhibition of nuclear targeting of PHLPP1 and DNMT1. BRAP2 protein persists late into spermiogenesis, being still present in the mature spermatozoan and hence able to interact with binding partners such as AKAP3 (yellow), which reach maximum levels in the mature spermatozoan. BRAP2 would appear to play a more structural/scaffold role to help localise proteins such as AKAP3 in the post acrosomal space and principal piece of sperm.

Techniques: Binding Assay, Expressing, Inhibition

Journal: PloS one

Article Title: Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response.

doi: 10.1371/journal.pone.0058911

Figure Lengend Snippet: Figure 1. Brap2 associates with Nedd8. (A) Schematic structure of the Nedd8 constructs for yeast two-hybrid screening. (B) Yeast PJ69-4A strains were transformed with expression vectors as indicated. Individual transformants were streaked to synthetic medium plates lacking tryptophan, leucine, histidine. (C) HEK293 cells were transfected with expression vectors as indicated and were subjected to immuno- precipitation (IP) with indicated antibodies. The total cell lysates and immunoprecipitants (IP) were subjected to immunoblot (IB) analyses with antibodies to Flag and HA. doi:10.1371/journal.pone.0058911.g001

Article Snippet: Antibodies and reagents Antibodies to Flag (M2, SIGMA), HA (A190-108A, Bethyl Lab., Inc), HA (Y-11, Santa Cruz), Ubiquitin (FK2, MBL), Brap2 (A302-682A, Bethyl Lab., Inc) were used for immunoblot analyses, PLOS ONE | www.plosone.org 8 March 2013 | Volume 8 | Issue 3 | e58911 HA (Y-11, Santa Cruz) and RelA/p65 (C22B4, Cell Signaling) for immunocytochemistry, and Anti-Flag M2-agarose and anti-HAagarose beads (SIGMA) for immunoprecipitation.

Techniques: Construct, Two Hybrid Screening, Transformation Assay, Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: PloS one

Article Title: Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response.

doi: 10.1371/journal.pone.0058911

Figure Lengend Snippet: Figure 2. The RING domain of Brap2 is responsible for the interaction with Nedd8. (A) Schematic structure of deletion mutants of Brap2. (B) HEK293 cells were transfected with indicated plasmids and were subjected to immunoprecipitation (IP). The immunoprecipitants (IP) and the total cell lysates were immunoblotted (IB) with indicated antibodies. (C) Schematic structure of point mutant of Brap2. (D) HEK293 cells were transfected with expression vectors as indicated and were subjected to IP. (E) HEK293 cells were transfected with HA-Nedd8 and Flag-Brap2, immunoprecipitated with Flag antibody. The total cell lysates and IP were immunoblotted with HA antibody. doi:10.1371/journal.pone.0058911.g002

Techniques: Transfection, Immunoprecipitation, Mutagenesis, Expressing

Journal: PloS one

Article Title: Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response.

doi: 10.1371/journal.pone.0058911

Figure Lengend Snippet: Figure 3. Brap2 is neddylated at lysine-432 in vivo. (A) Alignment of putative neddylation site of Brap2 with the consensus neddylation site of cullin family proteins. (B) HEK293 cells were transfected with indicated plasmids and were subjected to immunoprecipitation (IP) with Flag antibody and the resulting immunoprecipitant (IP) and total cell lysates were immnoblotted (IB) with indicated antibodies. doi:10.1371/journal.pone.0058911.g003

Techniques: In Vivo, Transfection, Immunoprecipitation

Journal: PloS one

Article Title: Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response.

doi: 10.1371/journal.pone.0058911

Figure Lengend Snippet: Figure 4. Brap2 dimerizes independent of Nedd8. (A) HEK293 cells were transfected with indicated plasmids and were then subjected to immunoprecipitation with Flag antibody. The total cell lysate and immunoprecipitant (IP) were immunoblotted (IB) using HA and Flag antibodies. Asterisks indicate non-specific bands. (B) HEK293 cells were transfected with indicated plasmids and incubated for 6 hours in the presence or absence of 5 mg/ml MLN4924, and were then subjected immunoprecipitation with HA antibody. The total cell lysate and immunoprecipitant (IP) were immunoblotted (IB) using HA and Flag antibodies. Asterisk indicates non-specific bands. doi:10.1371/journal.pone.0058911.g004

Techniques: Transfection, Immunoprecipitation, Incubation

Journal: PloS one

Article Title: Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response.

doi: 10.1371/journal.pone.0058911

Figure Lengend Snippet: Figure 5. Brap2 associates with Cul1 in a neddylation-independent manner. (A) HEK293 cells were transfected with HA-Cul1 and Flag-Brap2 WT or CA. HA-Cul1 was immunoprecipitated and total cell lysates and immunoprecipitants (IP) were immunoblotted (IB) with Flag and HA antibodies. (B) HEK293T cells were transfected with indicated plasmids and incubated for 1 hour in the presence or absence of 20 mM MG132, and subjected to immunoprecipitation (IP) analysis. Asterisk indicates non-specific bands. (C) HEK293T cells were transfected with indicated plasmids and incubated for 4 hours in the presence or absence of 1 mM MLN4924, and subjected to immunoprecipitation (IP). The immunoprecipitants (IP) and the total cell lysates were immunoblotted (IB) with indicated antibodies. doi:10.1371/journal.pone.0058911.g005

Techniques: Transfection, Immunoprecipitation, Incubation

Journal: PloS one

Article Title: Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response.

doi: 10.1371/journal.pone.0058911

Figure Lengend Snippet: Figure 6. Brap2 attenuates TNF-a-induced NF-kB translocation to nucleus. (A) HeLa cells were transfected with either Flag-Brap2 WT or Flag-Brap2 CA and stimulated with or without 5 ng/ml TNF-a for 30 min, and were subjected to immunocytochemistry using anti-Flag or anti-RelA/ p65 antibodies. Cells that express certain amount of Flag-Brap2 are marked by dot lines and control cells are marked by solid line. RelA/p65 translocates in nucleus after the stimulation. The right panels show the same cells using a rainbow color. Bars, 50 mm. (B) Ratiometric measurement of RelA/p65 fluorescence observed in cells expressing Flag-Brap2 before and after treatment with 5 ng/ml TNF-a (n = 50; mean 6 SEM; *** p,0.0001 by one-way ANOVA). (C) HEK293T cells were transfected with HA-Cul1 and Flag-Brap2 WT or CA, and stimulated with or without 5 ng/ml TNF-a for the indicated time, and subjected to immunoprecipitation (IP) with Flag antibody. The total cell lysates and IP were immunoblotted (IB) with indicated antibodies. (D) HEK293 cells were stimulated with 5 ng/ml TNF-a for indicated time, and cell lysates were subjected to immunoblot analysis. doi:10.1371/journal.pone.0058911.g006

Techniques: Translocation Assay, Transfection, Immunocytochemistry, Control, Fluorescence, Expressing, Immunoprecipitation, Western Blot

Journal: PloS one

Article Title: Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response.

doi: 10.1371/journal.pone.0058911

Figure Lengend Snippet: Figure 7. Neddylation of Brap2 is associated with TNF-a-induced NF-kB activity. (A) TNF-a-dependent activation of a NF-kB reporter gene in HEK293 cells expressing indicated plasmids. Cells were stimulated with 5 ng/ml TNF-a for 3 hours. (n = 3; mean 6 SEM; *** p,0.0001 by one-way ANOVA). (B) TNF-a-dependent activation of a NF-kB reporter gene in HEK293 cells expressing indicated plasmids. Cells were stimulated with 5 ng/ml TNF-a for 6 hours. (n = 3; mean 6 SEM; n.s., not significant by one-way ANOVA). doi:10.1371/journal.pone.0058911.g007

Techniques: Activity Assay, Activation Assay, Expressing

Journal: PloS one

Article Title: Brap2 regulates temporal control of NF-κB localization mediated by inflammatory response.

doi: 10.1371/journal.pone.0058911

Figure Lengend Snippet: Figure 8. Model of Brap2 function. (A) Model of Brap2 interaction with Cul1 and its effect on NF-kB signaling pathway. (B) Time course of NF-kB translocation and target gene expression by each Brap2 constructs. doi:10.1371/journal.pone.0058911.g008

Techniques: Translocation Assay, Targeted Gene Expression, Construct

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