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: Frontiers in Pharmacology

Article Title: PACSIN2 regulates mercaptopurine cytotoxicity and cellular biomechanics through Rac1 modulation in intestinal epithelial cells

doi: 10.3389/fphar.2026.1860702

Figure Lengend Snippet: The effect of PACSIN2 on mercaptopurine (MP) and the Rac1 inhibitor NSC23766 cytotoxicity. (A) Cytotoxicity MTT dose-response curve of LS180 MOCK, LS180 PACSIN2 KD cells, LS180 MOCK EMPTY, and LS180 MOCK PACSIN2 OVEREXPRESSION cells treated with MP (0.3 µM 160 µM) for 72 h. A significant difference of MP sensitivity between LS180 MOCK and LS180 PACSIN2 KD cells was detected at 0.3 µM (P = 0.001), 0.6 µM (P = 0.001), 1.25 µM (P = 0.0001), 2.5 µM (P = 0.0001). A significant difference of MP sensitivity between LS180 MOCK EMPTY and LS180 MOCK PACSIN2 cells was found at 1.25 µM (P < 0.05), 2.5 µM (P = 0.001), 5 µM (P = 0.0001), 10 µM (P = 0.001) and 20 µM (P = 0.01). Data are expressed as mean ± standard deviation from three independent experiments (Two-way ANOVA, post-test Tukey). (B) Cytotoxicity MTT dose-response curves of LS180 MOCK, LS180 PACSIN2 KD cells, LS180 MOCK EMPTY, and LS180 MOCK PACSIN2 OVEREXPRESSION cells treated with the Rac1 inhibitor NSC23766 (0.3 µM 160 µM) for 72 h. A significant difference from the Rac1 inhibitor NSC23766 was found between LS180 MOCK and KD cells (P < 0.0001)) and between LS180 EMPTY cells and LS180 PACSIN2 OVEREXPRESSION cells (P < 0.0001). Data are expressed as mean ± standard deviation from three independent experiments (Two-way ANOVA, post-test Tukey). (C) Cytotoxicity MTT dose-response curve LS180 MOCK, LS180 PACSIN2 KD cells, LS180 MOCK EMPTY and LS180 MOCK PACSIN2 OVEREXPRESSION cells co-treated with MP (from 0.3 µM to 160 µM) and the Rac1 inhibitor NSC23766 2.5 µM for 72 h. There was a significant difference between LS180 MOCK and LS180 KD cell lines (P < 0.0001)) and between LS180 MOCK EMPTY and LS180 MOCK PACSIN2 OVEREXPRESSION cell lines (P < 0.0001). Data are expressed as mean ± standard deviation from three independent experiments (Two-way ANOVA, post-test Tukey).

Article Snippet: The Rac1 G-LISA kit ( Cytoskeleton, Denver, CO, United States) was used to specifically detect active GTP-bound Rac1 and distinguish it from Rac2 and Rac3 isoforms in incubated cell/tissue lysates at a concentration of 1 mg/mL.

Techniques: Over Expression, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: PACSIN2 regulates mercaptopurine cytotoxicity and cellular biomechanics through Rac1 modulation in intestinal epithelial cells

doi: 10.3389/fphar.2026.1860702

Figure Lengend Snippet: The effect of PACSIN2 on Rac1 expression and activity levels. (A) Immunoblotting results of Rac1 protein levels in LS180 MOCK and LS180 PACSIN2 KD cells normalized against β‐actin (data expressed as mean ± standard deviation, n = 3, t‐test). (B) Rac1 protein activity evaluated as Rac1‐GTP amount in LS180 MOCK and LS180 PACSIN2 KD cell lines (data expressed as mean ± standard deviation, n = 3, t‐test). (C) immunoblotting results of Rac1 protein levels in LS180 MOCK cells transfected with an empty plasmid (LS180 EMPTY) used as control and LS180 cells presenting the PACSIN2 overexpression (LS180 PACSIN2 OVEREXPRESSION) cells at 48 after transfection (48 h) and after additional 72 h (72h post) normalized against β‐actin (data expressed as mean ± standard deviation, n = 3, t‐test). (D) Rac1 protein activity evaluated as Rac1‐GTP amount in LS180 EMPTY and LS180 PACSIN2 OVEREXPRESSION cells at 48 after transfection (48 h) and after additional 72 h (72h post, data expressed as mean ± standard deviation, n = 3, t‐test).

Article Snippet: The Rac1 G-LISA kit ( Cytoskeleton, Denver, CO, United States) was used to specifically detect active GTP-bound Rac1 and distinguish it from Rac2 and Rac3 isoforms in incubated cell/tissue lysates at a concentration of 1 mg/mL.

Techniques: Expressing, Activity Assay, Western Blot, Standard Deviation, Transfection, Plasmid Preparation, Control, Over Expression

Journal: Frontiers in Pharmacology

Article Title: PACSIN2 regulates mercaptopurine cytotoxicity and cellular biomechanics through Rac1 modulation in intestinal epithelial cells

doi: 10.3389/fphar.2026.1860702

Figure Lengend Snippet: Analysis of Rac1 protein levels under basal condition and after mercaptopurine (MP) treatment. Western Blot analysis of Rac1 protein levels in LS180 MOCK and LS180 PACSIN2 KD cell lines untreated and after 24 h (A) , 48 h (B) or 72 h (C) of MP treatment at the IC20, IC50 and IC80. Western Blot analysis of Rac1 protein levels in LS180 EMPTY and LS180 PACSIN2 OVEREXPRESSION cell lines untreated and after 24 h (D) , 48 h (E) or 72 h (F) of MP treatment at the IC20, IC50 and IC80. Rac1 levels were normalized using GAPDH or β‐actin and expressed in logarithmic scale (data expressed as mean ± standard deviation, n = 3, Two‐way ANOVA, post‐test Tukey). The β‐actin membranes of Panel (D–F) are the same presented in Panel (D–F) , respectively.

Article Snippet: The Rac1 G-LISA kit ( Cytoskeleton, Denver, CO, United States) was used to specifically detect active GTP-bound Rac1 and distinguish it from Rac2 and Rac3 isoforms in incubated cell/tissue lysates at a concentration of 1 mg/mL.

Techniques: Western Blot, Over Expression, Standard Deviation

Journal: Frontiers in Pharmacology

Article Title: PACSIN2 regulates mercaptopurine cytotoxicity and cellular biomechanics through Rac1 modulation in intestinal epithelial cells

doi: 10.3389/fphar.2026.1860702

Figure Lengend Snippet: Analysis of Rac1 protein activity after mercaptopurine (MP) treatment. G‐LISA analysis of Rac1 protein activity in LS180 MOCK and LS180 PACSIN2 KD cell lines untreated and after 24 h (A) , 48 h (B) or 72 h (C) of treatment at IC20, IC50 and IC80 of MP. G‐LISA analysis of Rac1 protein activity in LS180 EMPTY and LS180 PACSIN2 OVEREXPRESSION cell lines untreated and after 24 h (D) , 48 h (E) or 72 h (F) of treatment at IC20, IC50 and IC80 of MP (data expressed as mean ± standard deviation, n =3, Two‐way ANOVA, post‐test Tukey).

Article Snippet: The Rac1 G-LISA kit ( Cytoskeleton, Denver, CO, United States) was used to specifically detect active GTP-bound Rac1 and distinguish it from Rac2 and Rac3 isoforms in incubated cell/tissue lysates at a concentration of 1 mg/mL.

Techniques: Activity Assay, Over Expression, Standard Deviation