mouse anti ddk flag monoclonal antibody  (OriGene)

Journal: Nature

Article Title: Mitochondrial origins of the pressure to sleep

doi: 10.1038/s41586-025-09261-y

Figure Lengend Snippet: a , b , Volumetric renderings ( a ) and morphometric parameters ( b ) of automatically detected mitochondria in OPRM image stacks of dFBN dendrites in rested flies, sleep-deprived flies and flies allowed to recover for 24 h after sleep deprivation. Sleep history-dependent changes in mitochondrial number ( P < 0.0001, Holm–Šídák test after ANOVA), volume ( P = 0.0470, Dunn’s test after Kruskal–Wallis ANOVA), sphericity ( P = 0.0124, Dunn’s test after Kruskal–Wallis ANOVA) and branch length ( P = 0.0033, Dunn’s test after Kruskal–Wallis ANOVA) are occluded by the co-expression of AOX ( P ≥ 0.2257, two-sided t -test or Mann–Whitney test) or the simultaneous activation of TrpA1 ( P ≥ 0.0625, two-sided t -test or Mann–Whitney test) and (over)corrected after recovery sleep (number of mitochondria: P = 0.1551, all other parameters: P ≤ 0.0302, Dunn’s test after Kruskal–Wallis ANOVA). Two data points exceeding the y -axis limits are plotted as triangles at the top of the graphs; mean and s.e.m. are based on the actual values. c , Drp1 recruitment. Single confocal image planes through dFBN somata of flies expressing R23E10-GAL4 -driven mito-GFP (top) and Drp1 Flag from the endogenous locus (bottom). Sleep deprivation increases the percentage of cellular anti-Flag fluorescence (intensity-coded according to the key below) within automatically detected mitochondrial contours ( P < 0.0001, two-sided Mann–Whitney test). d , Mitochondria–endoplasmic reticulum contacts. Isosurface renderings (voxel value 128) of SPLICS puncta in distal dFBN dendritic branches (dashed outlines), obtained by trilinear interpolation of thresholded and despeckled confocal image stacks. Sleep deprivation increases the number of SPLICS puncta per dendritic field ( P < 0.0001, two-sided Mann–Whitney test). e , Mitophagy. Summed-intensity projections of dFBN dendrites expressing mito-QC. Emission ratios are intensity-coded according to the key below and increase after sleep deprivation ( P = 0.0101, two-sided t -test). Data are means ± s.e.m.; n , number of dendritic regions ( b , d , e ) or somata ( c ); asterisks, significant differences ( P < 0.05) in planned pairwise comparisons. Scale bars, 10 μm ( a ), 2 μm ( c ), 10 μm ( d ), 5 μm ( e ). For statistical details see Supplementary Table .

Article Snippet: Fixed brains were incubated sequentially at 4 °C in blocking solution (10% goat serum in 0.3% PBST) overnight, with mouse monoclonal anti-Flag antibody (anti-DDK; 1:1,000, OriGene) in blocking solution for 2–3 days, and with goat anti-Mouse Alexa Fluor 633 (1:500, ThermoFisher) in blocking solution for two days.

Techniques: Expressing, MANN-WHITNEY, Activation Assay, Fluorescence

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Exome Sequencing Reveals the Genetic Architecture of Non-syndromic Orofacial Clefts and Identifies BOC as a Novel Causal Gene.

doi: 10.1002/advs.202412073

Figure Lengend Snippet: Figure 6. BOC variants show defective cell surface localization and impaired binding with SHH and PTCH1. (A) p.R407W and p.R681X variants dislocate BOC from the cell surface to the cytoplasm. Hela cells transfected with HA-tagged WT or mutant BOC were processed for immunofluorescence analysis with an HA antibody. p.R407W and p.R681X BOC are diffused in the cytoplasm, while the WT BOC, as well as p.G436S and p.D1018N BOC, is localized on the membrane. Nuclear nucleus was stained with Hoechst. Scale bar, 20um. (B) p.R681X variant results in a secreted protein. HEK293T cells were transfected with WT or mutant BOC. 24 h later, cells were serum starved for 20 h. Cell lysate and concentrated medium were analyzed by Western blotting with an HA antibody. 𝛽-actin served as a loading control. (C) Effective secretion of SHH protein. The experiment was performed as in B, except that a Flag-tagged SHH construct was used. (D) p.R407W and p.R681X variants weaken the binding of BOC to SHH. Conditioned media of SHH-Flag was incubated with conditioned media of extracellular domains (ECD) of WT or mutant BOC, followed by a pull-down assay with a Flag antibody. The WT or mutant BOC-ECD protein bound on the beads was analyzed by Western blotting with an HA antibody. Note that the p.D1018N variant was not analyzed because of the localization of D1018 in the cytoplasmic tail, but not ECD. (E) p.R407W, p.G436S and p.D1018N variants dampen the binding to BOC to

Article Snippet: [30] The antibodies used were PTCH1 (CST, #2468S), HA (Proteintech, # 51064-2-AP), FLAG (Origene, #TA180144) and β-actin (Origene, #TA811000).

Techniques: Binding Assay, Transfection, Mutagenesis, Membrane, Staining, Variant Assay, Western Blot, Control, Construct, Incubation, Pull Down Assay