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Figure Lengend Snippet: Interactions between three closely related DUBs and a subset of RING E3 ligases. A , shown is a schematic representation of USP15, -4, and -11 domain structures. % indicates identity at amino acid level. B , shown is a summary of interactions between USP4, USP15, and USP11 (baits) and RING E3 ligases (preys) observed in a directed DUB:RING E3 ligase Y2H mating screen. Yeast diploids were scored after 5 days for growth using HIS3 and ADE2 reporters. The relative level of colony growth (+, ++, +++) is indicated by line thickness . Note that USP15 shares interactors with both USP4 and USP11. BRAP exclusively interacts with USP4 and USP15 among 55 DUBs tested in this screen. C , shown is a summary of the fragment-based mapping of the interaction between USP15 and BRAP described in .

Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-myc (4A6) (Millipore), polyclonal affinity-purified sheep anti-GFP (gift from Ian Prior, Liverpool, UK), mouse anti-FLAG (M2) (Sigma), rabbit anti-USP4 (A300–830A) and rabbit anti-USP15 (A300–923A), rabbit anti-BRAP (A302–681A and A302–682A) (Bethyl Laboratories), rabbit anti-CRAF (C20) (Santa Cruz), and rabbit anti-BRAP ( only ( )).

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Figure Lengend Snippet: USP15 is a positive regulator of MAP kinase signaling. USP15, but not USP4 depletion inhibits MEK and MAPK activation. A , HeLa cells were treated >72 h with 40 n m siRNA oligos against BRAP, USP4, USP15, USP4, and USP15 and KSR1. Cells were starved overnight in serum-free medium and stimulated with EGF (2 ng/ml, 30 min). MEK and MAPK (ERK) activation was assessed with phospho-specific antibodies (pMEK, pERK). CTRL , control. B and C , three individual siRNA oligos (#1, #2, #17) were used to deplete USP15 in HeLa cells, which were processed as in A . Shown are the relative phospho-MEK to total MEK levels in cells stimulated for 30 min with EGF, averaged over two (USP4, USP15-17) or three (all others) independent experiments carried out in duplicate ( error bars , S.E., two-tailed paired t test compared with control. B , USP15-1: p < 0.05, USP15-2 p < 0.05. C , USP15-1: p < 0.05, USP15-2: p < 0.01).

Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-myc (4A6) (Millipore), polyclonal affinity-purified sheep anti-GFP (gift from Ian Prior, Liverpool, UK), mouse anti-FLAG (M2) (Sigma), rabbit anti-USP4 (A300–830A) and rabbit anti-USP15 (A300–923A), rabbit anti-BRAP (A302–681A and A302–682A) (Bethyl Laboratories), rabbit anti-CRAF (C20) (Santa Cruz), and rabbit anti-BRAP ( only ( )).

Techniques: Activation Assay, Two Tailed Test

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Figure Lengend Snippet: siRNA-mediated depletion of USP15, but not USP4, destabilizes endogenous BRAP by promoting its ubiquitylation and proteasomal degradation. A , HeLa cells were treated with siRNA as indicated for 72 h before lysis. Lysates were probed with anti-USP15, BRAP, and actin antibodies. Quantitation shows the mean of three biological replicates ( error bars . S.E.; two-tailed paired t test compared with control; BRAP, p < 0.001; USP15-1, p < 0.005; USP15-17, p < 0.025). IB , immunoblot. B , HEK293T cells were either stimulated or not for 30 min with EGF. Lysates were subjected to immunoprecipitation ( IP ) with anti-BRAP and protein A-agarose beads or control beads, and samples were probed with mouse anti-USP15 and rabbit anti-BRAP antibodies. C , BRAP was immunoprecipitated from HeLa cells treated with either control reagents or USP15 siRNA (72 h) and lysed in RIPA buffer including 10 m m N -ethylmaleimide. Cell lysates and immunoprecipitation samples were probed with anti-ubiquitin, BRAP, USP15 antibodies ( Ab ). USP15 depletion promotes accumulation of a high molecular weight ubiquitylated species (*) of BRAP. D , proteasome inhibition rescues BRAP in USP15 depleted cells. HeLa cells were treated as in B for 66 h before incubation with 0.5 μ m epoxomicin or DMSO as a control for a further 8 h. Cells were lysed as in B , and lysates were probed with anti-BRAP. E , HeLa cells were treated with control reagents or USP15 siRNA (#17) for 66 h before cycloheximide ( CHX , 100 μg/ml) treatment. Cells were lysed at indicated time points, and lysates were probed as shown. * indicates high molecular weight BRAP species indicative of ubiquitylation. ( n = 3, error bars : S.E.). F , HEK293T cells were treated with control reagents or USP15 siRNA (#1) for 24 h before transfection with GFP or siRNA resistant forms of GFP-USP15 and GFP-USP15-C269S ( CS ) and further incubation for 48 h. Lysates were probed with anti-BRAP and USP15 ( light and darker exposures ).

Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-myc (4A6) (Millipore), polyclonal affinity-purified sheep anti-GFP (gift from Ian Prior, Liverpool, UK), mouse anti-FLAG (M2) (Sigma), rabbit anti-USP4 (A300–830A) and rabbit anti-USP15 (A300–923A), rabbit anti-BRAP (A302–681A and A302–682A) (Bethyl Laboratories), rabbit anti-CRAF (C20) (Santa Cruz), and rabbit anti-BRAP ( only ( )).

Techniques: Lysis, Quantitation Assay, Two Tailed Test, Western Blot, Immunoprecipitation, Molecular Weight, Inhibition, Incubation, Transfection

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Figure Lengend Snippet: The USP15 and USP4 DUSP-UBL mediates binding to the BRAP C-terminal coiled coil region Fragments of USP4, USP15, USP11, and BRAP were inserted into bait (USPs) and prey (BRAP) Y2H vectors and tested for interaction by directed Y2H. +, interaction seen with HIS3 reporter only; ++, interaction shown with HIS3 and ADE2 reporters. ND, not determined. Cat. domain, catalytic domain; ΔNT, N-terminal deletion mutant; CC, coiled coil.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-myc (4A6) (Millipore), polyclonal affinity-purified sheep anti-GFP (gift from Ian Prior, Liverpool, UK), mouse anti-FLAG (M2) (Sigma), rabbit anti-USP4 (A300–830A) and rabbit anti-USP15 (A300–923A), rabbit anti-BRAP (A302–681A and A302–682A) (Bethyl Laboratories), rabbit anti-CRAF (C20) (Santa Cruz), and rabbit anti-BRAP ( only ( )).

Techniques: Binding Assay, Mutagenesis

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Figure Lengend Snippet: BRAP interacts with both catalytically active and inactive USP4 and 15 but not USP11 in a cellular environment. HEK293T cells coexpressing myc-tagged BRAP and GFP-tagged USP4, -11, -15, GFP-BRAP- or GFP were lysed in RIPA buffer and subjected to immunoprecipitation ( IP ) with anti-GFP antibodies and probed with anti-myc and anti-GFP. Bound proteins were analyzed alongside 5% of input sample. IB , immunoblot. Arrows indicate higher molecular weight forms of BRAP. The bottom panel shows a higher intensity representation of input lanes. U , USP; CS , catalytically inactive mutant.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-myc (4A6) (Millipore), polyclonal affinity-purified sheep anti-GFP (gift from Ian Prior, Liverpool, UK), mouse anti-FLAG (M2) (Sigma), rabbit anti-USP4 (A300–830A) and rabbit anti-USP15 (A300–923A), rabbit anti-BRAP (A302–681A and A302–682A) (Bethyl Laboratories), rabbit anti-CRAF (C20) (Santa Cruz), and rabbit anti-BRAP ( only ( )).

Techniques: Immunoprecipitation, Western Blot, Molecular Weight, Mutagenesis

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Figure Lengend Snippet: Co-expression with catalytically inactive DUBs promotes the accumulation of ubiquitylated BRAP. HEK293T cells were transfected with FLAG-ubiquitin, GFP, or GFP-tagged active or inactive ( CS ) mutants of USP4 and -15 together with empty vector (vector) or myc-tagged BRAP or a mutant BRAP that is unable to recruit a ubiquitin conjugating E2 (WA). Cells were processed as described in , and immunoprecipitates ( IP ) were probed with anti-FLAG and anti-myc antibodies ( A ) or anti-GFP antibodies ( B ) to assess the ubiquitylation status of both BRAP ( A ) and USP4 and 15 ( B ). A , pink arrows indicate higher molecular weight bands reactive to anti-myc (BRAP) and anti-FLAG (Ub) antibodies in cells co-expressing wild-type BRAP ( wtBRAP ) and catalytically inactive USP4 ( U4 CS ) and USP15 ( U15 CS ). B , catalytically inactive USP15 and to a lesser extent also USP4 present with higher molecular weight bands that can be detected with FLAG-ubiquitin ( blue arrows ; see also ). U4 , USP4; U15 , USP15; CS , catalytically inactive mutants; WA , E2-binding deficient mutant. Lane 7 , M r marker. IB , immunoblot.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-myc (4A6) (Millipore), polyclonal affinity-purified sheep anti-GFP (gift from Ian Prior, Liverpool, UK), mouse anti-FLAG (M2) (Sigma), rabbit anti-USP4 (A300–830A) and rabbit anti-USP15 (A300–923A), rabbit anti-BRAP (A302–681A and A302–682A) (Bethyl Laboratories), rabbit anti-CRAF (C20) (Santa Cruz), and rabbit anti-BRAP ( only ( )).

Techniques: Expressing, Transfection, Plasmid Preparation, Mutagenesis, Molecular Weight, Binding Assay, Marker, Western Blot

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Figure Lengend Snippet: siRNA depletion of BRAP, but not USP15, stimulates MEK phosphorylation in non-starved cells stimulated with a short pulse of EGF. A , HeLa cells were treated for 72 h with siRNA targeting KSR1, BRAP, USP4, or USP15 (oligo #17) and serum-starved for the last 16 h before stimulation with EGF (1 ng/ml) for 5 min. Knockdown efficiency as well as MEK and ERK phosphorylation were assessed by Western blotting ( IB ). B , cells were treated as in A but were not serum-starved. Graphs show results from four biological replicates ( error bars , S.E., two-tailed paired t test compared with control; BRAP , p < 0.05; USP15: p < 0.01).

Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-myc (4A6) (Millipore), polyclonal affinity-purified sheep anti-GFP (gift from Ian Prior, Liverpool, UK), mouse anti-FLAG (M2) (Sigma), rabbit anti-USP4 (A300–830A) and rabbit anti-USP15 (A300–923A), rabbit anti-BRAP (A302–681A and A302–682A) (Bethyl Laboratories), rabbit anti-CRAF (C20) (Santa Cruz), and rabbit anti-BRAP ( only ( )).

Techniques: Western Blot, Two Tailed Test

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Figure Lengend Snippet: USP15 depletion in U2OS cells decreases CRAF expression levels and inhibits PDGF dependent pMEK activation. A , U2OS cells were treated >72 h with 20 n m of siRNA oligos targeting BRAP, USP4, and USP15 (two individual oligos). Cells were serum-starved for 16 h and then stimulated with 10 ng/ml PDGF for 5 min. RIPA lysates were probed with the indicated antibodies to assess knockdown efficiencies and expression levels or activation status of the key kinases of the MAPK cascade (CRAF, BRAF, MEK, and ERK). B , the ratio of pMEK/MEK, CRAF, and BRAP levels at 5 min post stimulation with PDGF, averaged from three independent experiments, are shown relative to control samples ( error bars , S.D.; two-tailed paired t test compared with control; pMEK-USP15-1, p < 0.05; USP15-2, p < 0.05; CRAF-USP15-1, p < 0.01; USP15-2: p < 0.05).

Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-myc (4A6) (Millipore), polyclonal affinity-purified sheep anti-GFP (gift from Ian Prior, Liverpool, UK), mouse anti-FLAG (M2) (Sigma), rabbit anti-USP4 (A300–830A) and rabbit anti-USP15 (A300–923A), rabbit anti-BRAP (A302–681A and A302–682A) (Bethyl Laboratories), rabbit anti-CRAF (C20) (Santa Cruz), and rabbit anti-BRAP ( only ( )).

Techniques: Expressing, Activation Assay, Two Tailed Test

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Figure Lengend Snippet: CRAF is the key target for USP15 regulation of ERK signaling. A , HeLa cells were treated with control reagents or CRAF siRNA for 60 h before starvation for 12 h. EGF (1 ng/ml) was added for various times before lysis. IB , immunoblot. B , direct comparison of the effect of USP15 and CRAF depletion in HeLa cells stimulated for 5 min with EGF ( n = 3, error bars ; S.E., paired two-tailed t test compared with 5 min; control, CRAF (5 min), p < 0.01; USP15 (5 min), p < 0.025; CRAF (10 min), p < 0.005; USP15 (10 min), p < 0.001). C , WM266-4 BRAF (V600D) expressing melanoma cells were treated with control reagents or target specific siRNA, and Nonidet P-40 cell lysates were probed with antibodies indicated. Depletion of USP15 by two individual oligos (#1 and #17) leads to reduced CRAF levels without affecting pMEK and pERK levels. * indicates a nonspecific band detected by the CRAF antibody. D , a working model is shown. i , USP15 positively controls signal output by regulating CRAF levels. ii , USP15 may also negatively regulate MAPK signaling by directly stabilizing BRAP.

Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-myc (4A6) (Millipore), polyclonal affinity-purified sheep anti-GFP (gift from Ian Prior, Liverpool, UK), mouse anti-FLAG (M2) (Sigma), rabbit anti-USP4 (A300–830A) and rabbit anti-USP15 (A300–923A), rabbit anti-BRAP (A302–681A and A302–682A) (Bethyl Laboratories), rabbit anti-CRAF (C20) (Santa Cruz), and rabbit anti-BRAP ( only ( )).

Techniques: Lysis, Western Blot, Two Tailed Test, Expressing

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

Journal: BMC Medical Genetics

Article Title: Lack of association between a functional variant of the BRCA-1 related associated protein ( BRAP ) gene and ischemic stroke

doi: 10.1186/1471-2350-14-17

Figure Lengend Snippet: The association between BRAP rs11066001 and stroke

Article Snippet: Genotyping of BRAP rs11066001 was conducted using the TaqMan genotyping assay (Applied Biosystems, Foster City, USA).

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