Journal: bioRxiv

Article Title: Red fluorescent labeling of myelin by membrane-targeted tdTomato in transgenic mouse lines

doi: 10.64898/2026.04.17.718425

Figure Lengend Snippet: Upper panels: Fluorescence micrographs visualizing that transgenically expressed tdTomato is localized in the processes and somata of oligodendrocytes which are marked by CNP immunolabeling. A fraction of CNP-positive oligodendrocytes does not express tdTomato (purple arrows), corresponding to the expression pattern as described in . Lower panels: Colocalization of MBP and tdTomato in myelin. The fluorescence signal is observed on both sides of the axon, while the enwrapped axon shows no fluorescence (white arrow in the inset). The cyan arrows highlight primary processes of oligodendrocytes connecting soma and myelin sheaths, which are labeled by tdTomato, but not stained for MBP. CB: cerebellum; CTX: cortex; HIP: hippocampus. The letters in the circles indicate the CNPMTO mouse line. Scale bar: 10 µm. Scale bar inset: 4 µm.

Article Snippet: Free-floating slices were permeabilized in 0.4% Triton X-100/PBS for 30 min and blocked with 4% fetal calf serum (FCS) in 0.2% Triton X-100/PBS for 1 h. Sections were immunohistochemically stained either for MBP (rat anti-MBP, AbD Serotec #MCA409S) or CNP (guinea pig anti-CNP, Synaptic Systems #355004).

Techniques: Fluorescence, Immunolabeling, Expressing, Labeling, Staining

Journal: bioRxiv

Article Title: Neprilysin inhibition reduces microtubule detyrosination in cardiomyocytes through a cGMP-PRKG1-VASH1 axis

doi: 10.64898/2026.03.13.711248

Figure Lengend Snippet: (A) Protocol: VASH1-KO hiPSC-CMs were kept in maturation medium for 2 weeks and then transduced for 6 days with lentivirus expressing YFP-tagged VASH1-WT, VASH1-7E or VASH1-7A each in combination with SVBP. (B) Representative Western blot of crude hiPSC-CM protein lysates stained for YFP-VASH1 and GAPDH (loading control) and quantification of VASH1/GAPDH in the 3 conditions (N/d=6/1). (C) Representative Western blot of crude hiPSC-CM protein lysates stained for YFP-VASH1, dTyr-tub, Tyr-tub and GAPDH (loading control) and quantification of dTyr-tub/GAPDH and Tyr-tub/GAPDH in non-transduced VASH1-KO hiPSC-CMs or transduced with YFP-VASH1-WT in combination with SVBP (N/d=6/1). (D) Representative Western blot of crude hiPSC-CM protein lysates stained for YFP-VASH1, dTyr-tub, Tyr-tub and GAPDH (loading control) and quantification of dTyr-tub/GAPDH and Tyr-tub/GAPDH in VASH1-KO hiPSC-CMs transduced with YFP-VASH1-7E or YFP-VASH1-7A in combination with SVBP (N/d=6/1). (E) Proposed model of regulation of VASH1 activity downstream of the action of sac. Sac inhibits neprilysin that would otherwise degrade the natriuretic peptides ANP, BNP and CNP. Through this inhibition, these natriuretic peptides can bind their respective receptors, which leads to the intracellular production of cGMP. Increased amounts of intracellular cGMP would then activate PRKG1A and thereby phosphorylate different targets, including VASH1. Eventually, phosphorylation of VASH1 reduces the binding of VASH1 to microtubules and tubulin detyrosination. Data are expressed as mean±SEM. Statistical significance was assessed with the one-way ANOVA and Tukey’s multiple comparisons tests (panel B) or unpaired Student’s t -test (panels C and D). Abbreviations: ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; cGMP, guanosine 3′,5′-cyclic monophosphate; CNP, C-type natriuretic peptide; dTyr-tub, detyrosinated tubulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GTP, guanosine triphosphate; hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes; KO, knock-out; MW, molecular weight marker; N/d, number of wells/differentiations; NPRA, natriuretic peptide receptor 1; NPRB, natriuretic peptide receptor 2; P, phosphorylated; pGC, particulate guanylate cyclase; PRKG1A, cGMP-dependent protein kinase G1 alpha; Tyr-tub, tyrosinated tubulin; VASH1, vasohibin 1; VASH1-7A, non-phosphorylatable VASH1 with 7 alanine residues; VASH1-7E, VASH1 phosphomimic with 7 glutamate residues; WT, wild-type.

Article Snippet: Cells were treated with 100 nM ET1 (Sigma Aldrich, 05-23-3800) alone or in combination with 40 μM sac (Sigma Aldrich, SML2064), 100 nM CNP (Tocris, 3520), or varying concentrations of ANP (Tocris, 1906), or RP8 (Tocris, 3028) at indicated concentrations.

Techniques: Expressing, Western Blot, Staining, Control, Transduction, Activity Assay, Inhibition, Phospho-proteomics, Binding Assay, Derivative Assay, Knock-Out, Molecular Weight, Marker