rhoa g lisa activation assay kit  (Cytoskeleton Inc)

Journal: Materials Today Bio

Article Title: Digestive-transformed titanium dioxide nanoparticles disrupt intestinal barrier via mitoxyperilysis mediated by mTOR-cytoskeleton axis dysregulation

doi: 10.1016/j.mtbio.2026.103674

Figure Lengend Snippet: Aberrant AKT/mTORC2 signaling axis activation mediates TiO 2 NPs-induced F-actin disassembly and plasma membrane injury. (a, b) Western blot (a) and densitometric quantification of the p -AKT/AKT ratio (b), confirming TiO 2 NPs-induced activation of the AKT/mTORC2 axis. (c) Quantification of RhoA expression, showing TiO 2 NPs-mediated suppression of RhoA-associated cytoskeletal regulation. (d) Representative confocal fluorescence images of F-actin stained with phalloidin (green) and DAPI (blue), visualizing the disruption of cytoskeletal architecture following TiO 2 NPs exposure. (e) Quantitative analysis of relative F-actin fluorescence intensity. (f, g) Western blot (f) and densitometric analysis of the p -AKT/AKT ratio (g), demonstrating the inhibitory effect of Torin-1 on TiO 2 NPs-induced AKT hyper-phosphorylation. (h) Quantification of F-actin fluorescence intensity, showing the restoration of cytoskeletal integrity by Torin-1 intervention. (i) Quantification of RhoA levels, indicating the rescue of TiO 2 NPs-suppressed RhoA by Torin-1. (j, k) CCK-8 cell viability (j) and LDH release (k) assays assessing the impact of Torin-1 on mitigating TiO 2 NPs-induced cytotoxicity and plasma membrane rupture. (l) Representative phalloidin-stained confocal images displaying the recovery of cytoskeletal morphology in the TiO 2 NPs + Torin-1 group. (m) Intracellular GSH content, revealing that Torin-1 intervention partially mitigates TiO 2 NPs-induced depletion of antioxidant reserves. Experimental groups: Control, Torin-1, TiO 2 NPs, and TiO 2 NPs + Torin-1. Data are presented as Mean ± SEM (n ≥ 3). Statistical significance is denoted by brackets and asterisks: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Intracellular RhoA activity was quantified utilizing the RhoA G-LISA Activation Assay Kit (Cytoskeleton; supplied by Jianglai Biotechnology, Shanghai, China; cat. no. CSK-BK124-S).

Techniques: Activation Assay, Clinical Proteomics, Membrane, Western Blot, Expressing, Cytoskeletal, Confocal, Fluorescence, Staining, Disruption, Analysis, Phospho-proteomics, CCK-8 Assay, Morphology, Control

Journal: Virulence

Article Title: Integrin β3 α2,6-sialylation by the sialyltransferase ST6Gal1 promotes human cytomegalovirus entry into fibroblasts

doi: 10.1080/21505594.2026.2732519

Figure Lengend Snippet: Integrin β3 sialylation participates in HCMV-induced cytoskeleton rearrangement during viral entry. (A) Integrin β3 sialylation defect resulted in downregulation of the Integrin β3-src signaling pathway. (B) The sialylation defect also resulted in impaired HCMV-induced RhoA inactivation, as shown by quantification. Data are presented as mean ± standard deviation ( n = 3), with **adjusted p < 0.01 vs The ST6Gal1 shRNA group, ***adjusted p < 0.001 vs The ST6Gal1 shRNA group. (C) Integrin β3 S85 sialylation was found to facilitate stress fiber disassembly and promote viral nuclear translocation. Representative immunofluorescence images for each group are shown (magnification: 600×, scale bar: 25 μm).

Article Snippet: Intracellular GTP-RhoA activity was determined using the G-LISA RhoA Activation Assay Biochem Kit (Cytoskeleton Inc., Cat#BK124, Denver, USA) according to the manufacturer’s instructions.

Techniques: Standard Deviation, shRNA, Translocation Assay, Immunofluorescence

Journal: bioRxiv

Article Title: Functional characterization of Rho GTPase activating proteins SYDE1 and SYDE2

doi: 10.64898/2026.08.31.748432

Figure Lengend Snippet: ( A ) Multiple turnover GAP activity assays with 500 nM SYDE1 and 5 μM RhoA, Rac1, or Cdc42. WT, wildtype; RA, R369A GAP-inactive mutant; SE, S578E phosphomimetic mutant. Data shown as mean ± SD from independent replicates (n ≥ 3). P values were calculated using ordinary one-way ANOVA with Tukey’s multiple comparisons test. ( B ) Multiple turnover GAP activity assays with 25 nM SYDE2 and 5 μM of RhoA, Rac1, or Cdc42. WT, wildtype; RA, R707A GAP-inactive mutant. Data shown as mean ± SD from independent replicates (n = 3). P values were calculated as for panel (A). ( C ) Multiple turnover GAP activity assays with 25 nM unphosphorylated (isolated from cells treated with JNK-IN-8 alone) or phosphorylated (isolated from cell treated with anisomycin) SYDE2 and 5 μM of RhoA, Rac1, or Cdc42. SYDE2 variants and significance calculations are as in panel (B). Bottom panel shows Phos-tag SDS-PAGE analysis to confirm phosphorylation states of SYDE2 used in the assays.

Article Snippet: GTP-bound GTPase levels in cell lysates were measured using G-LISA assay kits for RhoA (Cytoskeleton, Inc. #BK124), Rac1 (Cytoskeleton, Inc. #BK128), and Cdc42 (Cytoskeleton, Inc. #BK127).

Techniques: Activity Assay, Mutagenesis, Isolation, SDS Page, Phospho-proteomics

Journal: bioRxiv

Article Title: Functional characterization of Rho GTPase activating proteins SYDE1 and SYDE2

doi: 10.64898/2026.08.31.748432

Figure Lengend Snippet: ( A ) G-LISA effector binding assays for RhoA, Rac1, and Cdc42 in lysates from SYDE1 KO Swan 71 cells expressing the following constructs: GFP (−), SYDE1-GFP WT (WT), SYDE1-GFP R369A (RA), SYDE1-GFP S578E (SE). Data shown as mean ± SD from independent replicates (n = 5). All pairwise comparisons were insignificant (P >0.05) by ordinary one-way ANOVA with Tukey’s multiple comparisons test. ( B ) G-LISA effector interaction assays for RhoA, Rac1, and Cdc42 in lysates from HEK293 cells expressing GFP, SYDE1-GFP WT, RA, SE, and S578A (SA). Data shown as mean ± SD from independent replicates (n = 3). All pairwise comparisons were insignificant (P >0.05) by ordinary one-way ANOVA with Tukey’s multiple comparisons test. ( C ) G-LISA assays for RhoA, Rac1, and Cdc42 in lysates from HEK293 cells expressing GFP, SYDE2-GFP WT (WT), R707A (RA), S935D (SD), and S935A (SA). Data shown as mean ± SD from independent replicates (n = 3). All pairwise comparisons were insignificant (P >0.05) by ordinary one-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: GTP-bound GTPase levels in cell lysates were measured using G-LISA assay kits for RhoA (Cytoskeleton, Inc. #BK124), Rac1 (Cytoskeleton, Inc. #BK128), and Cdc42 (Cytoskeleton, Inc. #BK127).

Techniques: Binding Assay, Expressing, Construct

Journal: Bioactive Materials

Article Title: Mechanically sensitized hydrogel microspheres trigger membrane receptor switch for cartilage repair

doi: 10.1016/j.bioactmat.2026.03.017

Figure Lengend Snippet: Effect of OBNC microspheres on regulating MSCs. A) The expression of FAK and p-FAK detected by Western blot analysis. B) Quantitative analysis of the Western blot results. C) The expression of the integrin-α5 gene. D) The expression of the integrin-α8 gene. E) The expression of the Lamb-2 gene. F) The expression of the Spp-1 gene. G) The expression of the Vav-3 gene. H) The expression of the CDC-42 gene. I) Detection of the CDC-42 active protein by a GLISA kit. J) Detection of Rac-1 active protein by a GLISA kit. K) Detection of Rho A active protein by a GLISA kit. L) Mechanism of OBNC hydrogels triggering the membrane receptor switch and activating integrin receptors. (ns: non-significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001).

Article Snippet: RhoA GLISA Activation Assay (No. BK124, Cytoskeleton, USA), CDC42 GLISA Activation Assay (No. BK127, Cytoskeleton, USA), and Rac1 GLISA Activation Assay (No. BK128, Cytoskeleton, USA) were used to measure the activation of the GTPase protein family.

Techniques: Expressing, Western Blot, Membrane

Journal: Nature cell biology

Article Title: Elevated contractility drives age-associated implantation failure in mouse embryos from aged females

doi: 10.1038/s41556-026-02052-1

Figure Lengend Snippet: a) Schematic of contractile inhibitor pathways to regulate potential trophectoderm spreading behaviors. We hypothesized that RhoA activator CN03 would phenocopy aged embryos, while ROCK inhibitor Ripasudil would rescue aged spreading behaviors. b) Representative images of Young (left) and Young-CN03 treated (right) embryos fixed and stained for actin. Violin plot shows a reduction of spread area in Young-CN03 treated embryos (Young N = 9, n = 63 implanting blastocysts; Young -CN03 N = 5, n = 25 implanting blastocysts). Scale bar is 50μm. Two tailed p values from Mann-Whitney test. c) Representative images of Aged (left) and Aged Ripasudil-treated (right) embryos fixed and stained for actin. Violin plot shows a rescue of spread area in Aged Ripasudil-treated embryos (Aged N = 4, n = 27 implanting blastocysts; Aged Ripasudil N = 3, n = 16 implanting blastocysts). Scale bar is 50μm. Two tailed p values from Mann-Whitney test.

Article Snippet: Embryos were treated with equilibrated KSOM-AA and/or IVC media containing 1μg/mL RhoA Activator II (Cytoskeleton Cat #CN03) or 10μM Ripasudil (Selleck Chemical Cat#50-136-6407) or 10 uM ResEcad (Sigma-Aldrich Cat#205615).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature cell biology

Article Title: Elevated contractility drives age-associated implantation failure in mouse embryos from aged females

doi: 10.1038/s41556-026-02052-1

Figure Lengend Snippet: a) Schematic of trophectoderm spreading in which actomyosin generates contractile forces transmitted to other cells across E-cadherin–mediated junctions and to the substrate through integrin-based adhesions. b) Schematic of Trophectoderm Velocity Kymograph Analysis. TE contours were segmented at successive time points, and local displacements along the perimeter were used to compute edge velocities at each coordinate. These values were mapped over time to generate velocity kymographs, enabling spatial and temporal analyses of TE spreading. c) Embryos from aged mothers exhibit lower Trophectoderm Perimeter Velocity. Two tailed p values from Mann-Whitney Test. d) Perimeter outlines and Averaged trophectoderm velocity maps for Young (left) and Aged (right) conditions. Mural Trophectoderm perimeter outlines show the evolution of trophectoderm spreading over 24 hours. Perimeter outlines are color coded based on time post-implantation. Scale bar is 50 μm. Averaged TE Velocity Maps of normalized Trophectoderm contours for Young (n=20 implanting blastocysts) and Aged (n=15 implanting blastocysts). e) Schematic of pathway components contributing to tissue viscosity and reduced wetting behaviors. The cell-permeable drug CN03 activates RhoA for increased Myosin activity. Cell permeable ResEcad upregulates E-cadherin expression. Myosin-mediated forces at E-cadherin adhesions contribute to tissue viscosity and reduce tissue wetting. f) Quantification of Average Focal Adhesion Length per Embryo in Young (n=20 implanted embryos), Aged (n=17 implanted embryos), Young-CN03 treated (n=15 implanted embryos), and Young-ResEcad (n=12 implanted embryos) treated embryos (left). Two tailed p values from Mann-Whitney Test. Representative images of implanted embryos (right) demarcated with pMLC staining to show spread areas. Zoom images show localization of focal adhesion marker Paxillin at integrin adhesions. Scale bars are 50 μm and 5 μm, as indicated.

Article Snippet: Embryos were treated with equilibrated KSOM-AA and/or IVC media containing 1μg/mL RhoA Activator II (Cytoskeleton Cat #CN03) or 10μM Ripasudil (Selleck Chemical Cat#50-136-6407) or 10 uM ResEcad (Sigma-Aldrich Cat#205615).

Techniques: Two Tailed Test, MANN-WHITNEY, Viscosity, Activity Assay, Expressing, Staining, Marker