active rac1 pak 02  (Cytoskeleton Inc)

Journal: bioRxiv

Article Title: Sphingosine-1-phosphate cross-talks to Notch via a S1PR1-Dll4-MPDZ complex to regulate endothelial barrier function

doi: 10.64898/2026.05.20.726610

Figure Lengend Snippet: a , Immunoblots showing cleaved Notch1 (c-Notch1) and total Notch1 in MVECs treated with S1P for 1-hour ± γ-secretase inhibitor DAPT; quantification below (mean ± SEM). b , Fluorescent micrographs of YFP-Notch1 reporter intensity after S1P treatment; quantification below. Scale bar, 50 μm. c-d , Fold change in HES1 and HEY1 gene expression levels in (c) MVECs after S1P ± DAPT treatment, and in (d) mouse tissues after intravenous injection of S1P ± DAPT. e , Evans blue dye leakage in mouse dermis 1-hour post-injection with DMSO, S1P, or S1P + DAPT. Scale bar, 5 mm. f-i , Quantified dye leakage in (f) dermis, (g) lung, (h) liver, and (i) kidney, measured via absorbance. j , Heat maps of dextran dye diffusion in engineered microvessels ± S1P. Scale bar, 50 μm. k-l , Diffusive permeability in microvessels lined with (k) DAPT-vs. DMSO-treated or (l) NOTCH1 KO vs. SCR KO MVECs (mean ± SD). m , Micrographs of VE-cadherin (magenta), actin (green), and nuclei (blue) staining in microvessels. Scale bar, 50 μm. n-o , Mean corrected intensities of junctional (n) VE-cadherin and (o) actin from (m). p , Rac1 activity (PBD pull-down). Active/total Rac1 quantification below. q , Schematic of canonical Notch signaling. r , HES1 / HEY1 gene expression levels in dominant negative-MAML-GFP (DN-MAML-GFP) normalized to GFP expressing cells. s , Permeability in DN-MAML-GFP vs GFP-lined microvessels ± S1P. t , Schematic of Notch cortical pathway mediated by its transmembrane domain (TMD). u , Permeability in TMD-RFP vs RFP-lined microvessels ± S1P. v , Permeability in TMD-RFP vs RFP-lined microvessels ± clinical S1P receptor degrader fingolimod.

Article Snippet: PAK-PBD beads to bind active Rac1 (PAK-02) were purchased from Cytoskeleton and reconstituted according to the manufacturer’s instructions.

Techniques: Western Blot, Gene Expression, Injection, Diffusion-based Assay, Permeability, Staining, Activity Assay, Dominant Negative Mutation, Expressing

Journal: bioRxiv

Article Title: Toxin-triggered activation of regulated exocytosis enhances bacterial egress from the intestinal layer

doi: 10.64898/2026.02.06.704300

Figure Lengend Snippet: Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt), the YP147 (YPIII Δ cnfY ) +/- the empty vector pJNS11 (pV), and the cnfY + complementation plasmid pJNS10 (p cnfY ). ( A , D , E ) 3 hours post-infection the medium was removed and replaced by medium containing gentamicin and inhibitors of the Cdc42/Rac1 (ML141) ( A ), phospholipase C (PLC γ-1) (U-73122) ( D ), and Ca 2+ channel IP3-R (2-APB) ( D ), or activators for Cdc42/Rac1 (Activator II), PLC γ-1 (m-3M3FBS), or IP3-R (Myo-Inositol) ( E ). After 30 minutes, the medium from the basolateral chamber was collected and plated onto LB agar to determine the CFUs of egressed bacteria. The mean +/- SEM of three independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001). ( B , C ) Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt), the Yersinia virulence-negative strain (ΔpYV), the cnfY -negative mutant strain YP147 (Δ cnfY ) without or with cnfY + complementation plasmid pJNS10 (p cnfY ). After 3.5 h post-infection, infected Caco-2 cells were lysed, cell extracts were prepared. Activation of the different Rho GTPases was tested by the isolation of the GTP-bound form by pull-downs with Rho-GTPase-binding agarose beads and Western blotting using Rho GTPase-specific antibodies, e.g., against Cdc42. M: Protein size marker. As negative and positive controls, high concentrations of GDP and GTP (GTP γS) were added to the uninfected whole-cell extract samples. Equal concentrations of extracts were used for pull-down assays, which were assessed by Western blotting with Actin antibodies. ( B ) shows a scheme of the procedure (Created in BioRender. Dersch, P. (2026)), and ( C ) the Western blots; ( F , G ) CNF Y -mediated induction of inositol triphosphate (IP 3 ) production. ( F ) Scheme of triggered Cdc42-mediated activation of phospholipase C (PLC γ-1), which leads to the formation of inositol monophosphate (IP 3 ) from PIP 2. IP 3 is rapidly metabolized to inositol monophosphate (IP 1 ), and IP 1 can thus be used as a proxy for IP 3 levels by adding LiCl, which blocks the metabolism of IP 1 . Created in BioRender. Dersch, P. (2026). ( G ) Caco-2 monolayer was infected with 2.5 x 10 6 bacteria of the Y. pseudotuberculosis wildtype strain YPIII (wt), YP147 (YPIII Δ cnfY ) +/- the empty vector pJNS11 (pV), and the cnfY + complementation plasmid pJNS10 (p cnfY ). After at least 2 h post-infection, the medium was removed and replaced with medium +/- gentamicin and/or 50 mM LiCl to block IP 1 metabolism, as indicated. After an additional 1.5 h, the Caco-2 cells were lysed, and the cellular concentration of IP 1 was determined by ELISA using an anti-IP 1 monoclonal antibody. The IP 3 concentrations were calculated based on the IP 1 amounts. The mean +/- SEM of four independent biological replicates is shown. Significant differences were determined using a two-way ANOVA test with Tukey correction and indicated by asterisks (P-value: **** < 0.0001).

Article Snippet: After 2-4 hours post-infection, the media in the upper chamber were removed and replaced with a medium containing gentamicin (to kill extracellular bacteria on the apical side of the Caco-2 monolayer), with or without inhibitors/activators of cell signaling pathways/molecules with the following concentration of the inhibitors for RhoA (30 μM, Rhosin, Sigma-Aldrich, #555460), Rac-1/Cdc42 (2.5 μM ML141, MedChemExpress, #HY-12755), PLCγ-1 (5 μM, U-73122, MedChemExpress, #HY-13419), IP 3 -R (40 μM 2-APB, MedChemExpress, #HY-W009724), and the activators of Cdc42/Rac1 (1 unit/ml, Rac1/Cdc42 activator II, Cytoskeleton, Inc., #CN02-A), PLC γ-1 (15 μM m-3M3FBS, MedChemExpress, #HY-19619), and IP 3 -R (30 μM D-myo-Inositol-1,2,4,5-tetraphosphate, sodium salt, Santa Cruz Biotechnology, #sc-362076).

Techniques: Infection, Bacteria, Plasmid Preparation, Mutagenesis, Activation Assay, Isolation, Binding Assay, Western Blot, Marker, Blocking Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay