Journal: International Journal of Molecular Sciences

Article Title: cAMP and the Fibrous Sheath Protein CABYR (Ca 2+ -Binding Tyrosine-Phosphorylation-Regulated Protein) Is Required for 4D Sperm Movement

doi: 10.3390/ijms231810607

Figure Lengend Snippet: Cabyr–a Ca 2+ binding tyrosine-phosphorylation-regulated protein—is required for 4D sperm motility. ( A ) Computer-assisted sperm analysis (CASA) reveals decrease of motile sperm in Cabyr −/− mice (left), which correlates with a decrease in the number of sperm showing a progressive sperm movement (right). N = 4, n > 800 sperm. ( B ) Flagellar beat amplitude and beat frequency analyzed by CASA are not affected by Cabyr knockout. N = 4, n = 15. ( C ) Statistical analysis of curvilinear velocity by DHM. N = 4, n = 15. ( D ) Statistical analysis of lateral head displacement of Cabyr knockout in comparison to WT sperm. ( E ) Analysis of flagellar excursion in XY - and Z -plane in Cabyr −/− sperm shows a decrease of flagellar movement. N = 4, n = 15. ( F ) Representative 4D flagellar excursions of wt (left) and Cabyr −/− (right) sperm in which 3D flagellar excursions at different time points (0, 40, …, 280 ms) are color-coded and its projection onto the XY - and Z -plane are shadowed in black. * p < 0.05, ** p < 0.01, median: thick dashed lines; interquartile range: thin dashed lines.

Article Snippet: The Cabyr -KO (Ca 2+ -binding tyrosine-phosphorylation-regulated protein) animals were purchased from RIKEN BioResource Research Center (Kyoto, Japan).

Techniques: Binding Assay, Phospho-proteomics, Knock-Out, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Targeting the ASMase/S1P pathway protects from sortilin-evoked vascular damage in hypertension

doi: 10.1172/JCI146343

Figure Lengend Snippet: ( A ) Representative confocal immunofluorescence staining of Rac1 in HUVECs treated with vehicle (Ctrl) or sortilin alone or pretreated with the S1P3 inhibitor TY52156. Arrows indicate membrane translocation of Rac1. Scale bar: 10 μm. Insets show higher magnification, zoom ×4.7. ( B ) Acetylcholine-evoked vasorelaxation in WT mesenteric arteries exposed to vehicle, sortilin alone, or sortilin in the presence of NSC23766 ( n = 3). ( C ) Acetylcholine-evoked vasorelaxation in WT mesenteric arteries treated with vehicle or sortilin alone or pretreated with either ML171 or GSK2795039 before sortilin stimulation ( n = 3). ( D ) NOX activity in HUVECs treated with vehicle or sortilin alone or preincubated with ML171 or GSK2795039 before sortilin ( n = 4–5 replicates from 3 independent experiments). ( E ) NOX activity in WT and gp91 phox–/– mesenteric arteries exposed to vehicle or sortilin ( n = 3 replicates from 3 independent experiments). Data are expressed as increase of chemiluminescence per minute in arbitrary units. ( F ) Acetylcholine-evoked vasorelaxation in mesenteric arteries from WT and gp91 phox–/– mice exposed to vehicle or sortilin ( n = 5). ( G ) Representative immunoblots and densitometric analyses of 3 independent experiments evaluating protein levels of phospho-Tyr579-PYK2, phospho-Ser729-PKCɛ, phospho-Thr497-PKCα, PKC, S1P3, and Rac1-GTP in HUVECs treated with vehicle or sortilin in the presence or absence of TY52156 or GSK2795039. Data are represented as mean ± SD. One-way ANOVA ( D , E , and G ) or 2-way ANOVA ( B , C , and F ) followed by Bonferroni’s post hoc test was used. ( C ) * P < 0.0001 versus vehicle or GSK2795039 plus sortilin at the same acetylcholine concentration (as indicated by color code).

Article Snippet: Immunoblotting was performed as previously described , using the following antibodies: anti–phospho-eNOS serine 1177 (Enzo Life Sciences, catalog ALX-804-396-C100, clone 15E2); anti-phospho-eNOS-Thr494 (Cell Signaling Technology, catalog 9574); anti-eNOS (Cell Signaling Technology, catalog 9570); anti–β-actin (Abcam, mAb, catalog ab8226, clone mAbcam 8226); anti–phospho-protein-tyrosine kinase 2-β phospho-Tyr579 (PYK2B pY579) (Elabscience, catalog E-AB-21240); anti–phospho-PKCɛ Ser729 (Biorbyt, catalog orb315664); anti–PKCα (phospho T497) antibody (Abcam, catalog ab76016, clone EP2608Y); anti–PKC-PAN (Sigma-Aldrich, catalog SAB4502356); anti-ASMase (SMPD1; Biorbyt, catalog ORB214591); anti-S1P1 (Immunological Sciences, catalog AB-83739); anti-S1P3 (Elabscience, catalog E-AB-31267); anti-gp91phox (Santa Cruz Biotechnology Inc., catalog sc-130543, clone 54.1); anti-SphK1 (Santa Cruz Biotechnology Inc., catalog sc-365401, clone G-11); anti-SphK2 (MyBioSource, catalog MBS2518663); anti-aCDase (ASAH1; MyBioSource, catalog MBS1492517), and anti-active Rac1 (Rac1-GTP) (Neweast Bioscience, catalog 26903).

Techniques: Immunofluorescence, Staining, Translocation Assay, Activity Assay, Western Blot, Concentration Assay