Journal: Frontiers in Cellular Neuroscience

Article Title: The prion protein constitutively controls neuronal store-operated Ca 2+ entry through Fyn kinase

doi: 10.3389/fncel.2015.00416

Figure Lengend Snippet: The Tyr-kinase Fyn is the link between PrP C and SOCE. (A) PrP C reduces the level of active Fyn, as evident from both the representative WB (upper panel) of PrP-Tg and PrP-KO CGN probed with an antibody to p-SFK or total Fyn (both run in duplicate), under SOCE-activating conditions (Ca 2+ -depleted stores), and the corresponding densitometric analysis of p-SFK immunosignals normalized to that of total Fyn (lower panel). Contrary to other neuronal SFK members (Supplementary Figure 4), the identical apparent mass of p-SFK and Fyn indicates that the Fyn band corresponds to the p-SFK band. Similar results were obtained under basal conditions, i.e., with Ca 2+ -filled stores (see Supplementary Figure 3). (B,C) Addition (+) of the SFK inhibitors saracatinib (sara, 5 μM) and PP2 (10 μM), but not of PP3 (10 μM), reduces the level of both p-SFK (B) and total Tyr-phosphorylated (p-Tyr) proteins (C) of CGN compared to the untreated (−) samples. (B) The upper panel reports a representative WB of the two neuronal genotypes treated with, or without, the SFK inhibitors, and immunostained as in (A) , while the lower panel shows the corresponding densitometric analysis of p-SFK normalized to the band intensity of total Fyn. Veh (vehicle) indicates the control experiment run in the presence of DMSO (0.1%). (C) The upper panel reports the WB of total p-Tyr proteins present in the two CGN genotypes treated as in (B) . In the corresponding densitometric analysis (lowest panel), the p-Tyr band intensity was normalized to that of the Coomassie blue-stained bands (middle panel). (D) Only saracatinib and PP2 decrease SOCE-induced [Ca 2+ ] pm peaks, and abrogate the difference observed in control (veh-treated) PrP-Tg and PrP-KO CGN. (E) STIM1 is more abundantly Tyr-phosphorylated in PrP-KO CGN than in PrP-Tg CGN under SOCE-stimulating conditions. This is evident from the representative WB (left panel) of immunoprecipitated (IP) STIM1 probed with an antibody to either p-Tyr or total STIM1 (arrow), and from the corresponding densitometric analysis (right panel) reported as the ratio between the p-Tyr band intensity and the STIM1 band intensity. The arrowhead in the left panels indicates the immunosignal of the mouse IgG used in the immunoprecipitation assay. On the left of the WB, MW standards are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t -test. The analysis of the statistical significance ( p -value, Student’s t -test) of data for the comparison between different treatments within each PrP genotype (B–D) is reported in Supplementary Table 2. Other details are as in the legend to Figure .

Article Snippet: Instead, the SFK inhibitors [PP2 (10 μM, Tocris) and saracatinib (5 μM, Santa Cruz Biotechnology)], and the negative control of PP2, PP3 (10 μM, Santa Cruz Biotechnology), were added during the reconstitution step, and kept in the perfusion buffer throughout the entire experiment.

Techniques: Control, Staining, Immunoprecipitation, Comparison

Journal: bioRxiv

Article Title: Evaluating the impact of in silico predictors on clinical variant classification

doi: 10.1101/2021.08.09.455612

Figure Lengend Snippet: Application of PP3 and BP4 criteria to variants in this dataset.

Article Snippet: Collectively, the PP3 and BP4 criteria were applied by ClinGen VCEPs to 55% of missense variants in this data set.

Techniques:

Journal: bioRxiv

Article Title: Evaluating the impact of in silico predictors on clinical variant classification

doi: 10.1101/2021.08.09.455612

Figure Lengend Snippet: Effect of removing the PP3 and BP4 criteria on variants where in silico criteria were originally applied. (A) Removing PP3 caused 14% of pathogenic and 24% of likely pathogenic variants to downgrade to likely pathogenic and VUS, respectively. (B) Removing BP4 from likely benign variants caused 64% of these variants to move to a VUS classification.

Article Snippet: Collectively, the PP3 and BP4 criteria were applied by ClinGen VCEPs to 55% of missense variants in this data set.

Techniques: In Silico

Journal: bioRxiv

Article Title: Evaluating the impact of in silico predictors on clinical variant classification

doi: 10.1101/2021.08.09.455612

Figure Lengend Snippet: Point-based system for variant classification and the effect of removing in silico criteria when variants were evaluated using this approach. (A) Points awarded to benign and pathogenic evidence at distinct strength levels. (B) Total points required to reach pathogenic, likely pathogenic, VUS, likely benign, and benign classifications. (C) Effect of removing either PP3 or BP4 on variants that were classified using the point system and had in silico criteria applied originally.

Article Snippet: Collectively, the PP3 and BP4 criteria were applied by ClinGen VCEPs to 55% of missense variants in this data set.

Techniques: Variant Assay, In Silico

Journal: PLOS Genetics

Article Title: Leveraging cancer mutation data to inform the pathogenicity classification of germline missense variants

doi: 10.1371/journal.pgen.1011540

Figure Lengend Snippet: (A) The presence of either gain-of-function or loss-of-function mutations in cancer driver genes can lead to cancer (left) or rare Mendelian disorders (right) in different contexts. Most cancers result from somatic mutations that accumulate in a tissue-specific manner, whereas germline mutations are present in all cells of the body and cause a type of rare Mendelian disorder (e.g., neurodevelopmental disorder). (B) The HRAS Q61K mutation is an example of a known cancer mutation that drives different types of cancers that also causes Costello syndrome, a developmental disorder, when observed as a germline variant. (C) Workflow for extracting cancer mutations from Cancer Hotspots. Recurrent cancer mutations were filtered to 2,447 missense mutations. See main text for details. REVEL scores thresholds correspond to supporting evidence for pathogenicity (PP3) and for benign-ness (BP4). Created with Lucidchart. Created with BioRender.

Article Snippet: We grouped these CH mutations by REVEL scores using the ClinGen-proposed PP3/BP4 score thresholds ( ) [ ].

Techniques: Mutagenesis, Variant Assay