cdc42 activator  (Cytoskeleton Inc)

Journal: iScience

Article Title: TLR4-paxillin-Rac1 signaling mediates LPS-induced dysregulation of keratinocyte function

doi: 10.1016/j.isci.2026.115943

Figure Lengend Snippet: Rac1/paxillin axis mediates LPS-induced keratinocyte migration (A and B) Representative immunoblots and corresponding quantification of phosphorylated levels of paxillin at Ser273 ( p -paxillin Ser273 ) and total paxillin in keratinocytes pretreated with the Rac1 inhibitor NSC23766 (50 μM) for 30 min prior to stimulation with LPS (100 ng/mL) for 15 min. LPS significantly increased Ser273 phosphorylation, which was abolished by Rac1 inhibition. LPS increases Ser273 phosphorylation, which is abrogated by Rac1 inhibition. (C and D) Representative western blot and corresponding quantification of p -paxillin Ser273 and total paxillin in keratinocytes treated with Rac/Cdc42 activator II (0.25 units/mL) for 15, 30, and 60 min. Total paxillin and β-actin served as loading controls. p -paxillin Ser273 increased in a time-dependent manner, with peak induction observed at 15–30 min following Rac/Cdc42 activation. (E and F) Representative immunoblots and corresponding quantification of active Rac1 (Rac1-GTPase) in keratinocytes expressing wild-type paxillin (Pax WT), non-phosphorylatable paxillin (Pax A, S273A), or phosphomimetic paxillin (Pax D, S273D), treated with or without LPS (100 ng/mL) for 15 min. LPS increased Rac1 activation in Pax WT and Pax D cells, whereas Rac1 activation was attenuated in Pax A expressing cells. Data are represented as mean ± SD. N = 3, n = 3. Statistical analysis was performed using one-way ANOVA. Significant differences: ∗, p value < 0.05, ∗∗, p value < 0.01, ∗∗∗, p value < 0.001, ns, not significant.

Article Snippet: Rac/Cdc42 Activator , Cytoskeleton , Cat#CN02.

Techniques: Migration, Western Blot, Phospho-proteomics, Inhibition, Activation Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: Hypothalamic arcuate microglia in sepsis: AgRP circuit engagement and ARHGAP24-dependent remodeling

doi: 10.1186/s12974-026-03786-3

Figure Lengend Snippet: Rac1/Cdc42 signaling regulates microglial morphology and modifies outcomes after CLP. A Rhodamine phalloidin staining of F-actin in BV2 cells treated with DMSO, AZA1, LPS, or LPS+AZA1 in shCtrl and sh- Arhgap24 (1#) cells. Scale bar, 5 μm. B and C Quantification of filopodia length ( B ) and filopodia number per cell ( C ) from ( A ). N = 8 to 12 cells per condition. D BrdU staining of BV2 cells treated with DMSO or AZA1 in shCtrl and sh- Arhgap24 (1#) groups. Scale bar, 50 μm. E Quantification of BrdU positive cells from ( D ). N = 5 to 6. F Rhodamine phalloidin staining of F-actin in BV2 cells 5 min after CN02 treatment in shCtrl and sh-Arhgap24 (1#) groups. Scale bar, 5 μm. G and H Quantification of filopodia length ( G ) and filopodia number per cell ( H ) from ( F ). N = 5 to 6 cells per condition. I Experimental design for daily third ventricle administration of aCSF, AZA1, or CN02 from day 0 to day 7 after CLP, with analyses at CLP1d and CLP7d. J Representative Iba1 immunostaining in ARC sections at CLP1d after aCSF, AZA1, or CN02 treatment, with higher magnification views below. K Quantification of Iba1 positive cell counts per ARC section from ( J ). N = 5. L to P Sholl analysis ( L ) and quantification of microglial morphological parameters in the ARC, including Sholl area under the curve ( M ), maximal Sholl radius ( N ), total process length ( O ), and mean process diameter ( P ). N = 4 to 6. Q Kaplan Meier survival curves with restricted mean survival time (RMST) indicated. N = 8 to 10. R Clinical disease scores at CLP1d and CLP7d. N = 8 to 10 at CLP1d and N = 3 to 6 at CLP7d. S to U Serum IL-1β ( S ), TNF-α ( T ), and IL-6 ( U ) levels at CLP7d. V Serum corticosterone levels at CLP7d. N = 3 to 6. Data are shown as mean ± SEM. For ( B , C , G , H ), unpaired two tailed Student’s t test. For ( E , K , M to P , R ), one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. ns, not significant

Article Snippet: AZA1 (TargetMol, 100 μg/mouse/day) and CN02 (Cytoskeleton, 0.4 U/mouse/day) were delivered as single or daily doses according to the design.

Techniques: Staining, BrdU Staining, Immunostaining, Two Tailed Test

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