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: Scientific Reports

Article Title: Intrinsic Photosensitivity Enhances Motility of T Lymphocytes

doi: 10.1038/srep39479

Figure Lengend Snippet: ( a ) Summary of Ca 2+ responses evoked by blue light (4.7 mW cm −2 , 450 mJ cm −2 ) in Jurkat cells pretreated (see Methods) with U73122 (500 nM), U733433 (5 μM), wortmanin (1 μM), genestein (100 μM), PP2 (3 μM), PP3 (3 μM), CD45 inhibitor (1 μM), ascorbic acid (10 mM), DHA (docosahexaenoic acid, 5 μM), apocynin (100 μM), antimycin/rotenone/oligomycin (20: 20:5 μM), DPI (diphenyliodonium, 50 μM) (n = 30–40 cells per group in triplicate), *P < 0.001. ( b and c ) Blue light dose-dependently increases phosphorylation of Lck(Y394) in Jurkat cells detected with a generic pSrc (Y416) antibody (see ), data are mean of 3 experiments. The Src inhibitor, PP2, blocks the increase in pY394 demonstrating that light triggers trans autophosphorylation of Lck. ( d ) Blue light increases pSrc in murine CD3 + T cells. ( e ) Blue light triggers phosphorylation of ZAP-70 (Y319) in Jurkat cells. ( f ) White light (13.7 J cm −2 ) stimulates tyrosine phosphorylation (Y783) of PLC-γ1. Note that the immunoblots are cropped and the full blots are shown in . ( g–i ) Mean changes in Fluo4 fluorescence and cumulative activation plots in response to blue light for wild-type Jurkat cells, PLC-γ1-deficient Jurkat T cells (Jgamma1), Jgamma1 cells stably expressing PLC-γ1 (JgammaWT), Lck-deficient Jurkat cells (JCam1.6) and cells lacking TCRβ expression (n = 40–120 for each group). ( j ) Light-evoked Ca 2+ responses in control and Ca 2+ -free media; n = 40–50. The rising phase of responses is aligned to show the time course of decay.

Article Snippet: PP2 and PP3 were from Tocris.

Techniques: Phospho-proteomics, Western Blot, Fluorescence, Activation Assay, Stable Transfection, Expressing, Control

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