ihc test of brca1 protein expression  (Human Protein Atlas)

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: Anti-β-Actin antibody (sc-47778, Santa Cruz) was used as loading control and for quantification of relative BRCA1 protein expression levels.

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: Anti-β-Actin antibody (sc-47778, Santa Cruz) was used as loading control and for quantification of relative BRCA1 protein expression levels.

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: Anti-β-Actin antibody (sc-47778, Santa Cruz) was used as loading control and for quantification of relative BRCA1 protein expression levels.

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: Anti-β-Actin antibody (sc-47778, Santa Cruz) was used as loading control and for quantification of relative BRCA1 protein expression levels.

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: Anti-β-Actin antibody (sc-47778, Santa Cruz) was used as loading control and for quantification of relative BRCA1 protein expression levels.

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