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: (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.

Article Snippet: 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, HA (Y-11, Santa Cruz) and RelA/p65 (C22B4, Cell Signaling) for immunocytochemistry, and Anti-Flag M2-agarose and anti-HA-agarose beads (SIGMA) for immunoprecipitation.

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: (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.

Article Snippet: 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, HA (Y-11, Santa Cruz) and RelA/p65 (C22B4, Cell Signaling) for immunocytochemistry, and Anti-Flag M2-agarose and anti-HA-agarose beads (SIGMA) for immunoprecipitation.

Techniques: 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: (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 µM 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 µM MLN4924, and subjected to immunoprecipitation (IP). The immunoprecipitants (IP) and the total cell lysates were immunoblotted (IB) with indicated antibodies.

Article Snippet: 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, HA (Y-11, Santa Cruz) and RelA/p65 (C22B4, Cell Signaling) for immunocytochemistry, and Anti-Flag M2-agarose and anti-HA-agarose beads (SIGMA) for immunoprecipitation.

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: (A) HeLa cells were transfected with either Flag-Brap2 WT or Flag-Brap2 CA and stimulated with or without 5 ng/ml TNF-α 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 µm. ( B ) Ratiometric measurement of RelA/p65 fluorescence observed in cells expressing Flag-Brap2 before and after treatment with 5 ng/ml TNF-α (n = 50; mean ± 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-α 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-α for indicated time, and cell lysates were subjected to immunoblot analysis.

Article Snippet: 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, HA (Y-11, Santa Cruz) and RelA/p65 (C22B4, Cell Signaling) for immunocytochemistry, and Anti-Flag M2-agarose and anti-HA-agarose beads (SIGMA) for immunoprecipitation.

Techniques: 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: (A) Model of Brap2 interaction with Cul1 and its effect on NF-κB signaling pathway. ( B ) Time course of NF-κB translocation and target gene expression by each Brap2 constructs.

Article Snippet: 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, HA (Y-11, Santa Cruz) and RelA/p65 (C22B4, Cell Signaling) for immunocytochemistry, and Anti-Flag M2-agarose and anti-HA-agarose beads (SIGMA) for immunoprecipitation.

Techniques: Translocation Assay, Targeted Gene Expression, Construct

Journal: Cancers

Article Title: Mutation of PTPN11 (Encoding SHP-2) Promotes MEK Activation and Malignant Progression in Neurofibromin-Deficient Cells in a Manner Sensitive to BRAP Mutation

doi: 10.3390/cancers14102377

Figure Lengend Snippet: Relation of phenotypes induced by BRAP and PTPN11 missense mutations to NF1 inactivation. Immunoblot analysis was performed for SHP-2, BRAP , neurofibromin, as well as phosphorylated and total forms of Akt, ERK, and MEK in parental HeLa cells as well as HeLa cells transiently expressing SHP-2 (G503V) or BRAP (G370A). The uncropped western blot figures are shown in . ( A ) or in the corresponding stably infected cells transfected with control or NF1 siRNAs for 48 h ( B ). sNF96.2-GFP cells expressing SHP-2 (G503V) and BRAP (G370A) are shown for comparison to indicate the cleavage product of SHP-2 (arrowhead). The uncropped western blot figures are shown in .

Article Snippet: Immunoblot analysis was performed as previously described [ ] with primary antibodies to phosphorylated Akt (#4060), to Akt (#9272), to phosphorylated ERK1/2 (#4370), to ERK1/2 (#9102), to phosphorylated MEK (#9121), to MEK (#9122), to phosphorylated S6 (#4858), to S6 (#2217), to phosphorylated STAT3 (#9131), to STAT3 (#4904), and to SHP-2 (#3752), all of which were obtained from Cell Signaling Technology, as well as with those to BRAP (sc-166012), to neurofibromin (sc-376886), and to α-tubulin (sc-32293) from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Stable Transfection, Infection, Transfection, Control, Comparison