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: NPJ Regenerative Medicine

Article Title: Novel copper-ion coordinated andrographolide-loaded hydrogel activates Rac1/JNK1 axis for enhancing diabetic wound healing

doi: 10.1038/s41536-026-00457-y

Figure Lengend Snippet: A Venny plots of Andro target genes vs DWs causative genes. B PPI network highlighting key signaling proteins modulated by Andro treatment. Nodes represent individual proteins (colored by pathway/function; see legend); edges represent validated interactions. Major pathway clusters are annotated (e.g., JNK1/MAPK, Rac1/cell migration). C GO functional enrichment analysis of intersecting targets, showing significantly enriched biological processes (e.g., inflammatory response, cell migration, angiogenesis) ranked by gene count or significance. D KEGG pathway enrichment analysis ranked by gene count and significance. E Top 20 KEGG pathway enrichment analysis (sorted by enrichment score). F Representative docking pose of Andro within the proteins binding pocket, showing key noncovalent interactions.

Article Snippet: Rac1 protein (MCE, China) was diluted to 50 μg/mL in sodium acetate and immobilized onto the chip’s second channel at a flow rate of 10 μL/min.

Techniques: Migration, Functional Assay, Binding Assay

Journal: NPJ Regenerative Medicine

Article Title: Novel copper-ion coordinated andrographolide-loaded hydrogel activates Rac1/JNK1 axis for enhancing diabetic wound healing

doi: 10.1038/s41536-026-00457-y

Figure Lengend Snippet: A RMSD of complex, protein, and Ligand, reflecting structural deviation and conformational stability over time. B Rg of the complex, indicating overall compactness and structural tightness. C Rac1 and Andro binding site distance, monitoring binding stability and positional consistency. D FEL showing a single dominant basin, indicating conformational stability without large-scale reorganization. E Buried area between Andro and Rac1, representing the extent of buried surface and interaction strength. F Simulated conformational superposition, illustrating structural flexibility and binding site variations. G Modeling trajectory change, displaying dynamic shifts in binding site and molecular orientation. H PCA and protein structure, highlighting dominant motion modes and conformational flexibility. I Andro and Rac1 binding energies VDW and ELE, showing contributions from van der Waals and electrostatic interactions. J Hydrogen bond number, reflecting the stability and strength of polar interactions. K Hydrogen bond frequency between Andro and Rac1, indicating persistence and key residues involved in H-bond formation.

Article Snippet: Rac1 protein (MCE, China) was diluted to 50 μg/mL in sodium acetate and immobilized onto the chip’s second channel at a flow rate of 10 μL/min.

Techniques: Binding Assay

Journal: NPJ Regenerative Medicine

Article Title: Novel copper-ion coordinated andrographolide-loaded hydrogel activates Rac1/JNK1 axis for enhancing diabetic wound healing

doi: 10.1038/s41536-026-00457-y

Figure Lengend Snippet: A , B Representative western blots ( A ) and quantification ( B ) of Rac1, JNK1, Jun, and Fos protein levels in wound tissues at day 3 for each treatment group. C Relative mRNA expression levels of Rac1 , Jnk1 , Jun , and Fos in day-3 DW tissues. D SPR analysis confirming direct binding of Andro to Rac1 with a dissociation constant (KD) of 3.82 µM. E Relative mRNA expression levels of Rac1 , Jnk1 , Jun , and Fos in day-7 wound tissues. F , G Representative western blots ( F ) and quantification ( G ) of Rac1, JNK1, Jun, and Fos protein levels in day-7 wound tissues. The bar charts present mean ± SD ( n = 3 per group). * P < 0.05, ** P < 0.01, *** P < 0.001, vs Control group, # P < 0.05, ## P < 0.01, ### P < 0.001, vs Matrix group. H Schematic model summarizing the proposed mechanism by which ASFH-delivered Andro treats DWs. Andro directly binds Rac1, activates the Rac1/JNK1/Jun/Fos signaling axis, promotes M1-to-M2 macrophage polarization, modulates cytokine profiles, enhances angiogenesis and collagen remodeling, and collectively accelerates wound closure.

Article Snippet: Rac1 protein (MCE, China) was diluted to 50 μg/mL in sodium acetate and immobilized onto the chip’s second channel at a flow rate of 10 μL/min.

Techniques: Western Blot, Expressing, Binding Assay, Control

Journal: Cell reports

Article Title: The STRIPAK complex is required for radial sorting and laminin receptor expression in Schwann cells

doi: 10.1016/j.celrep.2025.115401

Figure Lengend Snippet: (A and B) Glutathione S-transferase (GST) pull-down from P5 wild-type (WT) mouse sciatic nerve lysate, comparing GST-Rac1-GDP versus GST-Rac1-GTP (A) Volcano plot showing the Welch difference (log2 fold change) of Rac1 interactors. Proteins in red, such as STRN3 and several well-established Rac1 interactors, are significantly (false discovery rate [FDR] = 0.05) enriched in the active Rac1-GTP fraction compared to the inactive Rac1-GDP fraction. MOB4 was found to be enriched in the Rac1-GTP fraction in a separate biological replicate (data not shown). (B) GST-Rac1-GDP/GTP pull-down of P5 WT sciatic nerve lysate was repeated and analyzed by western blot (WB). STRN3 was observed to elute with the active Rac1-GTP fraction but not the inactive Rac1-GDP fraction. (C) Immunoprecipitation of Rac1 from SC lysates. STRN3 was found to interact with Rac1 in SCs. n = 5 individual coIPs per targeting antibody (anti-Rac1 or IgG isotype control). Unpaired two-tailed t test ( t = 3.730, df = 8, p = 0.0058). Error bars indicate SEM. ** p < 0.01.

Article Snippet: For each charge reaction, bait proteins His-Rac1 (Cytoskeleton, Inc. RC01-XL) or His-human serum albumin (His-HSA, Abcam ab217817) negative control were diluted to 10 mM in 140 mL PD6 buffer (5 mM Tris-HCl pH 7.4, 12.5 mM NaCl 2 , 0.25 mM MgCl 2 , and 1:400 protease inhibitor cocktail) containing 1 mM GDP or GTPgS and incubated for 20 min at 37°C.

Techniques: Western Blot, Immunoprecipitation, Control, Two Tailed Test

Journal: Cell reports

Article Title: The STRIPAK complex is required for radial sorting and laminin receptor expression in Schwann cells

doi: 10.1016/j.celrep.2025.115401

Figure Lengend Snippet: (A) Representative western blots of Rac1 and CDC42 active (GTP) and total (tot) forms from pooled P20 sciatic nerves and brachial plexuses of WT and Strn1/3 dSCKO mice. (B and C) Densitometry analysis shows no significant change in Rac1 or CDC42 activity in peripheral nerves of Strn1/3 dSCKO mice at P20. n =5 samples per genotype. Samples were pooled from 3–5 animals each. Unpaired two-tailed t test. Rac1 activity ( t = 1.649, df = 8, p = 0.1379) and CDC42 activity ( t = 1.257, df = 8, p = 0.2443). (D) Representative western blots of PAK1 and NF2 phosphorylated and tot forms from P20 sciatic nerves and brachial plexuses of WT and Strn1/ 3 dSCKO mice. (E and F) Densitometry analysis shows no significant change in phosphorylation or tot levels of the Rac1/CDC42 effectors PAK1 or NF2 in peripheral nerves of Strn1/3 dSCKO mice at P20. n = 4 animals per genotype. Unpaired two-tailed t test. p-PAK1 ( t = 1.484, df = 6, p = 0.1884), PAK1 ( t = 0.5307, df = 6, p = 0.6147), p-NF2 ( t = 1.160, df = 6, p = 0.2900), and NF2 ( t = 0.7988, df = 6, p = 0.4548). Error bars indicate SEM.

Article Snippet: For each charge reaction, bait proteins His-Rac1 (Cytoskeleton, Inc. RC01-XL) or His-human serum albumin (His-HSA, Abcam ab217817) negative control were diluted to 10 mM in 140 mL PD6 buffer (5 mM Tris-HCl pH 7.4, 12.5 mM NaCl 2 , 0.25 mM MgCl 2 , and 1:400 protease inhibitor cocktail) containing 1 mM GDP or GTPgS and incubated for 20 min at 37°C.

Techniques: Western Blot, Activity Assay, Two Tailed Test

Journal: Cell reports

Article Title: The STRIPAK complex is required for radial sorting and laminin receptor expression in Schwann cells

doi: 10.1016/j.celrep.2025.115401

Figure Lengend Snippet:

Article Snippet: For each charge reaction, bait proteins His-Rac1 (Cytoskeleton, Inc. RC01-XL) or His-human serum albumin (His-HSA, Abcam ab217817) negative control were diluted to 10 mM in 140 mL PD6 buffer (5 mM Tris-HCl pH 7.4, 12.5 mM NaCl 2 , 0.25 mM MgCl 2 , and 1:400 protease inhibitor cocktail) containing 1 mM GDP or GTPgS and incubated for 20 min at 37°C.

Techniques: Generated, Virus, Recombinant, SYBR Green Assay, Protease Inhibitor, Plasmid Preparation, In Situ, BIA-KA, Mutagenesis, Software

Journal: Cell reports

Article Title: The STRIPAK complex is required for radial sorting and laminin receptor expression in Schwann cells

doi: 10.1016/j.celrep.2025.115401

Figure Lengend Snippet:

Article Snippet: His-Rac1 , Cytoskeleton, Inc. , RC01-XL.

Techniques: Generated, Virus, Recombinant, SYBR Green Assay, Protease Inhibitor, Plasmid Preparation, In Situ, BIA-KA, Mutagenesis, Software

Journal: Cell reports

Article Title: The STRIPAK complex is required for radial sorting and laminin receptor expression in Schwann cells

doi: 10.1016/j.celrep.2025.115401

Figure Lengend Snippet:

Article Snippet: GST-Rac1 , Cytoskeleton, Inc. , RCG01-C.

Techniques: Generated, Virus, Recombinant, SYBR Green Assay, Protease Inhibitor, Plasmid Preparation, In Situ, BIA-KA, Mutagenesis, Software