Journal: PLoS ONE

Article Title: Differential Regulation of PDE5 Expression in Left and Right Ventricles of Feline Hypertrophy Models

doi: 10.1371/journal.pone.0019922

Figure Lengend Snippet: (a) Similarity between feline PDE5 and PDE5s from other species. The feline PDE5 sequence was obtain from the translation of the cloned cDNA sequence. Sequences of PDE5 from other species were obtained from GenBank. Multi-sequence alignments were calculated using Clustal W. The % identities between feline and other species are in bold. (b) Domain structure of the feline PDE5 protein. GAF indicates cGMP binding domain, and HDc the phosphohydrolase catalytic domain. Structure prediction was performed using the SMART software. (c) Alignment of protein sequences from the N-terminal region of feline and human PDE5 variants. The shaded area indicates identical residues and the residues in feline PDE5 that are different from those of human variants are noted with asterisks. The start codons are in bold faces and amino acid residue numbers are indicated.

Article Snippet: Rabbit-polyclonal antibody against human PDE5 was from Cell Signaling (Danvers, MA) and rabbit-polyclonal antibodies against cGKI and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Sequencing, Clone Assay, Binding Assay, Software, Residue

Journal: PLoS ONE

Article Title: Differential Regulation of PDE5 Expression in Left and Right Ventricles of Feline Hypertrophy Models

doi: 10.1371/journal.pone.0019922

Figure Lengend Snippet: (a) Immunoblot analysis and coomassie blue staining of whole cell extracts made from E. coli cells carrying feline PDE5 expression plasmid pT7-PDE5 or a control vector pSNAP-tag(T7). Lanes 1 and 3 are extracts from cells carrying pSNAP-tag(T7). Lanes 2 and 4 are extracts from cells carrying pT7-PDE5. Lanes 1 and 2 are Coomassie blue staining of the gel. Lanes 3 and 4 are immunoblot using an anti-human PDE5 antibody. (b) Measurement of PDE5 activity. PDE activity of the extracts from cells carrying pT7-PDE5 in the presence or absence of a PDE5 inhibitor, MY5445, was shown (see Method for details). Basal level PDE activity determined with extract from cells carrying pSNAP-tag(T7) was subtracted. The error bars indicate standard deviation (n = 4).

Article Snippet: Rabbit-polyclonal antibody against human PDE5 was from Cell Signaling (Danvers, MA) and rabbit-polyclonal antibodies against cGKI and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Western Blot, Staining, Expressing, Plasmid Preparation, Control, Activity Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Differential Regulation of PDE5 Expression in Left and Right Ventricles of Feline Hypertrophy Models

doi: 10.1371/journal.pone.0019922

Figure Lengend Snippet: Consecutive paraffin embedded RV tissue sections from control feline hearts were used to examine PDE5 expression. The section in a) was incubated first with a rabbit polyclonal anti-PDE5 antibody followed by HRP conjugated anti-rabbit secondary antibody and then developed using ImmPACT NovaRED as a substrate to visualize the bonded antibodies. Section b) was processed in parallel with a) but omitted the primary anti-PDE5 antibody. Hematoxylin QS was used as nuclear counterstaining.

Article Snippet: Rabbit-polyclonal antibody against human PDE5 was from Cell Signaling (Danvers, MA) and rabbit-polyclonal antibodies against cGKI and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Expressing, Incubation

Journal: PLoS ONE

Article Title: Differential Regulation of PDE5 Expression in Left and Right Ventricles of Feline Hypertrophy Models

doi: 10.1371/journal.pone.0019922

Figure Lengend Snippet: Tissue homogenates from RV and LV of control animals were prepared as described in . cGMP specific PDE activity in these samples were measured in the presence or absence of 120 uM MY5445, a specific inhibitor of PDE5, as described in . The error bars indicate standard deviation (n = 4).

Article Snippet: Rabbit-polyclonal antibody against human PDE5 was from Cell Signaling (Danvers, MA) and rabbit-polyclonal antibodies against cGKI and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Activity Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Differential Regulation of PDE5 Expression in Left and Right Ventricles of Feline Hypertrophy Models

doi: 10.1371/journal.pone.0019922

Figure Lengend Snippet: Tissue homogenates from LV and RV of control, PA-banded and Ao-banded animal models were analyzed for PDE5 expression by immunoblotting. (a) Homogenates from LV samples. (b) Homogenate from RV samples. Samples from control, PA-banded and Ao-banded groups were indicated in the figure. E. coli extracts from strains carrying pT7-PDE5 and pSNAP-tag(T7) were used as positive and negative controls (shown as+and -). GAPDH levels in the same set of tissue samples were also determined by immunoblotting, and used for normalization of PDE5 levels. (c) Normalized average PDE level in LV tissues from each group (n = 4). (d) Normalized average PDE level in RV tissues from each group. The error bars indicate standard deviations. Student t-test p values that show statistical significance are indicated in the figure.

Article Snippet: Rabbit-polyclonal antibody against human PDE5 was from Cell Signaling (Danvers, MA) and rabbit-polyclonal antibodies against cGKI and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Control, Expressing, Western Blot

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