Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) MCF7 and MDA-MB-231 cells were cultured for either 48 hours in 10% charcoal stripped serum (CSS) or 24 hours in serum starved (0.5%) conditions. Control cells were cultured in normal growth medium (10% fetal bovine serum). Cell lysates (100 μg) were immunoblotted for BRCA1, pS473 AKT, and pan AKT. pS473 AKT indicates activation of AKT; pan AKT serves as a loading control. Molecular weights (kD) are indicated. The upper band in the BRCA1 panel for MCF7 cells is the full length (p220) BRCA1 protein. The lower band may represent a splice variant that is not consistently observed across experimental replicates or conditions.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Cell Culture, Control, Activation Assay, Variant Assay

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) MCF7 cells cultured in normal growth medium were treated with the indicated doses of LY294002 for 18 hours and then harvested for immunoblot analysis. Controls include both untreated cells and DMSO vehicle treated cells. Molecular weights (kD) are indicated. The immunoblot was probed with rabbit polyclonal phospho-AKT-substrate antibody (PAS), then stripped and reprobed with a mouse monoclonal BRCA1 antibody. An immunoreactive band was observed (*) of the same molecular weight with both antibodies which decreased in a dose-dependent manner upon LY294002 treatment. Immunoblots for BARD1, pS473 AKT, and pan AKT are presented in the third panel. The pS473 AKT antibody bound to a cross-reactive band of lower molecular weight in this experiment which was not recognized by the pan AKT antibody.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Cell Culture, Western Blot, Molecular Weight

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) Full length, purified BRCA1 protein (250 ng/lane) was added to an in vitro kinase assay plus or minus active AKT1 kinase. GST and GST-GSK3 were included as negative and positive controls respectively. Molecular weights (kD) are indicated. Reaction products were separated by SDS-PAGE and 32P-labelled products were detected by exposure to a phosphor screen. Silver stain demonstrated equal loading of BRCA1 (*) in the experimental lanes. The predominant band in all lanes is BSA (a component of the enzyme dilution buffer).

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Purification, In Vitro, Kinase Assay, SDS Page, Silver Staining

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) Whole cell extracts were prepared from MCF7 cells and separated by SDS-PAGE. After transfer, the PVDF membrane was split longitudinally and each lane was incubated separately with the indicated antibodies: mouse monoclonal anti-BRCA1 (Ab4) and rabbit polyclonal anti-pS694 BRCA1 (at dilutions of 1:500 and 1:1000). Additionally, pS694 BRCA1 antibody at a dilution of 1:500 was incubated with the membrane either without or with pre-incubation with the specific phosphorylated antigen matrix (lanes four and five respectively) as a control for specificity of the antibody. Separately incubated lanes (1-3 and 4-5) were reassembled and exposed to the same piece of film for chemiluminescent detection. Molecular weights (kD) are indicated.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: SDS Page, Membrane, Incubation, Control

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) MCF7 cells cultured in 10% CSS medium were stimulated with 10 nM E2 or pretreated with 10 μM LY294002 for 1 hour before stimulation with E2 for indicated amount of time (in hours). Immunoblot for BRCA1 and BARD1 proteins demonstrates that pre-incubation with LY294002 blocks the increase in total protein levels observed with E2 treatment alone. Samples treated with LY294002 alone showed similar levels of BRCA1 and BARD1 expression as the E2 + LY294002 groups (data not shown). pS473-AKT was detected to monitor AKT activation. Numbers below each panel represent relative densitometry values compared to the vehicle control.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Cell Culture, Western Blot, Incubation, Expressing, Activation Assay, Control

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: (A) MCF7 cells were transduced with the indicated adenoviral vectors, cultured for 48 hours in CSS medium, then fixed and stained with both an N-terminal mouse monoclonal BRCA1 antibody (red) and an exon 11 directed rabbit polyclonal BRCA1 antibody (green). Yellow indicates overlap of both signals to demonstrate specific staining for BRCA1. Nuclei were counterstained with DAPI. The following MOI's were used: 100 for Ad-myr-AKT, Ad-kd-AKT, and control Ad-LacZ; 25 for Ad-BRCA1.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Transduction, Cell Culture, Staining, Control

Journal: Molecular and cellular endocrinology

Article Title: AKT Regulates BRCA1 Stability in Response to Hormone Signaling

doi: 10.1016/j.mce.2010.01.019

Figure Lengend Snippet: MCF7 cells were transduced with the indicated adenoviral vectors and plated at low density in triplicate in six-well plates. Cells were cultured in CSS medium for 24 hours prior to irradiation with indicated doses and maintained in CSS medium for an additional 48 hours following irradiation. Cells were then switched into normal growth medium to allow for colony outgrowth. Survival is presented on a logarithmic scale as the number of remaining colonies at each dose divided by the average number of colonies in unirradiated wells of the same transduction group. The following MOI's were used: 100 for Ad-myr-AKT, Ad-kd-AKT, and control Ad-GFP; 10 for Ad-BRCA1 and Ad-BR1853. Error bars represent SEM, N=3. A student's t-test was performed for BRCA1/myr-AKT vs. mock, GFP, myr-AKT, BRCA1/GFP, or BR1853/myr-AKT and found to be significant at p≤ 0.01 for both 2 and 4 Gy treatment groups (*) in all cases.

Article Snippet: Purified full length BRCA1 protein was obtained from ProteinOne. pGEX-BRCA1 S694/T696 fusion protein was phosphorylated by AKT1 and prepared for mass spectrometry as previously described ( 69 ).

Techniques: Transduction, Cell Culture, Irradiation, Control

Journal: Brain sciences

Article Title: Colocalization of BRCA1 with Tau Aggregates in Human Tauopathies.

doi: 10.3390/brainsci10010007

Figure Lengend Snippet: Figure 1. Immunohistochemical studies using antibodies against phosphorylated tau (AT8) and BRCA1 (MS110) in human tauopathy patients’ brains. (A–D) Neurofibrillary tangles (NFTs) and neuropil threads in Alzheimer’s disease (AD) patients’ brains were immunopositive for BRCA1 (E,F). In normal control (NC), BRCA1 positivity was observed in the nucleus in the hippocampus despite AT8 positivity in the cytoplasm. (G,H) Pick bodies in Pick’s disease (PiD) were immunopositive for BRCA1. (I–L) Globose NFT in the midbrain and precentral cortex and glial coiled bodies in the cerebral white matter of progressive supranuclear palsy (PSP) patients’ brains were immunopositive for BRCA1. (M–P) Although some BRCA1 positivity partially resembling tuft-shaped astrocytes was observed in the frontal cortex (M,N), cytoplasmic BRCA1 positivity was not observed close to most of the AT8-positive tuft-shaped astrocytes in PSP patients (O,P). (Q–T) In corticobasal degeneration (CBD) patients’ brains, no cytoplasmic BRCA1 staining was observed close to pretangles or astrocytic plaques. p-tau, phosphorylated tau. The scale bars represent 20 µm.

Article Snippet: The human full-length BRCA1 protein-coding sequence was amplified by polymerase chain reaction (PCR) using pBABEpuro HA Brca1 plasmid (gift from Prof. Stephen Elledge, Addgene plasmid # 14999) [48] as a template.

Techniques: Immunohistochemical staining, Control, Staining

Journal: Brain sciences

Article Title: Colocalization of BRCA1 with Tau Aggregates in Human Tauopathies.

doi: 10.3390/brainsci10010007

Figure Lengend Snippet: Figure 2. Immunofluorescence double staining of human tauopathy patients’ brains using antibodies against phosphorylated tau (AT8) and BRCA1 (MS110). Colocalization of p-tau and BRCA1 was observed in NFTs of the AD brain samples (A), Pick bodies of the PiD brain samples (C), and globose NFTs (D), glial coiled bodies (E), and some of the tuft-shaped astrocytes in the PSP brain samples (F) but not in the NC brain samples (B). The scale bars represent 20 µm.

Article Snippet: The human full-length BRCA1 protein-coding sequence was amplified by polymerase chain reaction (PCR) using pBABEpuro HA Brca1 plasmid (gift from Prof. Stephen Elledge, Addgene plasmid # 14999) [48] as a template.

Techniques: Double Staining

Journal: Brain sciences

Article Title: Colocalization of BRCA1 with Tau Aggregates in Human Tauopathies.

doi: 10.3390/brainsci10010007

Figure Lengend Snippet: Figure 3. Immunohistochemical studies using anti-BRCA1 antibodies in disease controls. BRCA1 positivity resembling phosphorylated α-synuclein (psyn) or phosphorylated TDP-43 (pTDP-43) positive inclusions was not observed in dementia with Lewy bodies (DLB), Parkinson’s disease with dementia (PDD), multiple system atrophy cerebellar type (MSA-C), or amyotrophic lateral sclerosis (ALS). The scale bar represents 20 µm.

Article Snippet: The human full-length BRCA1 protein-coding sequence was amplified by polymerase chain reaction (PCR) using pBABEpuro HA Brca1 plasmid (gift from Prof. Stephen Elledge, Addgene plasmid # 14999) [48] as a template.

Techniques: Immunohistochemical staining

Journal: Brain sciences

Article Title: Colocalization of BRCA1 with Tau Aggregates in Human Tauopathies.

doi: 10.3390/brainsci10010007

Figure Lengend Snippet: Figure 4. Western blot analyses of the sarkosyl fractionated human brain samples. (A) Sarkosyl-soluble fraction of brain samples showed similar intensity bands of full-length BRCA1 (≈300 kDa) among the AD, NC, and PSP brain samples, with high intensity stacking observed in 2 AD brain samples and 1 PSP midbrain sample. In the sarkosyl-insoluble fraction, tau bands were only observed in AD and PSP brain samples. Sarkosyl-insoluble full-length BRCA1 (≈300 kDa) was observed in both AD samples as previously described. Although sarkosyl-insoluble full-length BRCA1 monomer band intensities in PSP samples were higher than in the NC in only 1 midbrain sample, the stacking intensities were higher in PSP samples, which suggests BRCA1 aggregation. T: temporal cortex, PCG: precentral gyrus, MB: midbrain. (B) Quantification of sarkosyl-insoluble BRCA1 signal at ≈300 kDa. Intensity relative to the average intensity of AD samples is shown in box and whisker plots. Although much less than AD, PSP brain samples showed significantly more insoluble BRCA1 than NC.

Article Snippet: The human full-length BRCA1 protein-coding sequence was amplified by polymerase chain reaction (PCR) using pBABEpuro HA Brca1 plasmid (gift from Prof. Stephen Elledge, Addgene plasmid # 14999) [48] as a template.

Techniques: Western Blot, Whisker Assay

Journal: Brain sciences

Article Title: Colocalization of BRCA1 with Tau Aggregates in Human Tauopathies.

doi: 10.3390/brainsci10010007

Figure Lengend Snippet: Figure 5. Western blot of HEK293FT cells overexpressing Venus-tagged human full-length BRCA1 with or without transfection with P301L tau and in vitro generated tau seeds. Sarkosyl-insoluble BRCA1 was observed only in the cell sample transfected with both P301L tau plasmid and tau seeds, which showed sarkosyl-insoluble tau.

Article Snippet: The human full-length BRCA1 protein-coding sequence was amplified by polymerase chain reaction (PCR) using pBABEpuro HA Brca1 plasmid (gift from Prof. Stephen Elledge, Addgene plasmid # 14999) [48] as a template.

Techniques: Western Blot, Transfection, In Vitro, Generated, Plasmid Preparation

Journal: Brain sciences

Article Title: Colocalization of BRCA1 with Tau Aggregates in Human Tauopathies.

doi: 10.3390/brainsci10010007

Figure Lengend Snippet: Figure 6. Immunohistochemical (A) and immunofluorescence double staining (B) studies using antibodies against BRCA1 and BARD1 in human tauopathies. (A) Cytoplasmic BARD1 positivity resembling tau aggregates was not observed in AD, PiD, or PSP brain samples. (B) BARD1 did not colocalize with BRCA1 in the cytoplasm and showed weak positivity in the nucleus. NFT, neurofibrillary tangle. The scale bars represent 20 µm in both A and B.

Article Snippet: The human full-length BRCA1 protein-coding sequence was amplified by polymerase chain reaction (PCR) using pBABEpuro HA Brca1 plasmid (gift from Prof. Stephen Elledge, Addgene plasmid # 14999) [48] as a template.

Techniques: Immunohistochemical staining, Double Staining

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 1. Schematic presentation of BRCA1 and location of the investigated missense variants. RING = Really Interesting New Gene, NES = Nuclear Export Signal, NLS = Nuclear Localisation Signal, BRCT = BRCA1 C-terminal. Figure adapted from [8].

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques:

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 2. Protein expression levels of BRCA1 variants determined by Western blot analysis: HEK293FT cells were transiently transfected with BRCA1 WT, known benign and pathogenic control variants and 14 missense BRCA1 VUSs. The black dots represent individual normalised band intensi- ties. Each column represents the mean of three to six independent replicates (n = 3–6). The benign (green) and pathogenic (orange) control variants are grouped to the left. Variants marked with a red * indicate p < 0.05. Error bars represent standard deviation.

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques: Expressing, Western Blot, Transfection, Control, Standard Deviation

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 3. mRNA levels of BRCA1 variants in HEK293FT cells determined by qPCR: HEK293FT cells were transfected with plasmids encoding BRCA1 WT and the four variants found to be expressed at protein levels lower or similar to the included pathogenic controls, as shown in Figure 2. Each column represents the mean of three or four independent replicates (n = 3–4), and the black dots represent individual values after normalisation using actin. Error bars represent standard deviation.

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques: Transfection, Standard Deviation

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 5. Assessment of BRCA1 protein variant stability after 8 h by cycloheximide chase assay: HEK293FT cells were transiently transfected with BRCA1 WT, known benign and pathogenic control variants and 11 missense BRCA1 VUSs. The columns show normalised mean protein levels of three to five independent replicates (n = 3–5) after 8 h of treatment with cycloheximide relative to the levels at 0 h of treatment (100%) for each individual variant. The black dots represent individual normalised band intensities. Error bars represent standard deviation. The benign and pathogenic control variants are coloured green and orange, respectively. Variants marked with a red * indicate a significant reduction in protein stability compared with WT protein (p < 0.05).

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques: Variant Assay, Transfection, Control, Standard Deviation

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 6. Assessment of protein interactions between BRCA1 and BARD1 or PALB2 by Co-IP assay: (A) HEK293FT cells were transiently co-transfected with EV or BRCA1 construct together with Flag- PALB2. Cells were harvested 48 h post transfection and co-immunoprecipitation (Co-IP) was performed. Input = input cell lysates, Co-IP = eluates from the Flag-column. BRCA1 (220 kDa) was detected with anti-BRCA1. PALB2-Flag (130 kDa) was detected with anti-Flag. Representative results from one of in total three experiments are shown. (B) Identical experiment to (A), with BARD1-V5 and V5 antibody coupled to the magnetic beads. BARD1-V5 (100 kDa) was detected with anti-V5. (C) Quantified results from BRCA1-PALB2 Co-IP. Western blot bands from three biological replicates were quantified by Image Lab software (n = 3). Black dots represent individual normalised band intensities. Graphs represent mean % compared to the WT. Error bars represent standard deviation. The benign (green) and pathogenic (orange) control variants are grouped to the left. In the initial analysis, the variant p.Lys503Arg appeared to have a reduced binding to PALB2, but this interaction was shown to be similar to the WT/benign controls when quantifying against the amount of the variant input samplemarked by a red ∧). (D) Identical experiment to (C), but with BRCA1-BARD1 Co-IP.

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques: Co-Immunoprecipitation Assay, Transfection, Construct, Immunoprecipitation, Magnetic Beads, Western Blot, Software, Standard Deviation, Control, Variant Assay, Binding Assay