gtp bound rac1  (Cytoskeleton Inc)

Journal: Cell Death Discovery

Article Title: Hydrogen sulfide protects against spinal cord pyroptosis via persulfidation of Rac1 after lumbosacral plexus nerve injury

doi: 10.1038/s41420-025-02736-x

Figure Lengend Snippet: A Principal component analysis (PCA) plot showing significant clustering differences in gene expression between two groups. B Heatmap of differentially expressed genes (DEGs). C Volcano plot identifying 294 upregulated and 2074 downregulated genes. D Sample correlation heatmap clarifying relationships among samples. E , F GO enrichment analysis showing significant terms. G KEGG enrichment analysis highlighting pathways like NF-κB. H Western blot confirming Rac1 gene silencing via lentiviral transfection. I qRT-PCR results reveal that H₂S suppressed NLRP3, Caspase-1, and GSDMD expression in hypoxia-treated neurons, but this inhibitory effect was reduced when Rac1 was silenced. n = 3, * P < 0.05, ** P < 0.01. L Light microscopy images of each group. Scale bar, 50 μm.

Article Snippet: Active Rac1 (GTP-bound) levels were quantified using the G-LISA® Activation Assay Kit (Cytoskeleton, CO, USA) following manufacturer guidelines.

Techniques: Gene Expression, Western Blot, Transfection, Quantitative RT-PCR, Expressing, Light Microscopy

Journal: Cell Death Discovery

Article Title: Hydrogen sulfide protects against spinal cord pyroptosis via persulfidation of Rac1 after lumbosacral plexus nerve injury

doi: 10.1038/s41420-025-02736-x

Figure Lengend Snippet: A , B qRT-PCR and Western blot analyses reveal that H₂S does not regulate the transcriptional expression or protein level of Rac1 in neuronal cells. C H₂S intervention inhibits Rac1 activity in neurons, detected via immunoprecipitation and Western blot for Rac1-GTP. D The ratio of NADPH to NADP⁺ in neuronal cells was measured across groups. n = 3, * P < 0.05, ** P < 0.01. E ROS levels in neuronal cells were quantified for each group. n = 3, * P < 0.05, ** P < 0.01. F Sulfhydration of purified Rac1 protein. n = 3, * P < 0.05, ** P < 0.01. G – J Western blot and quantitative analysis reveal that pretreatment with DTT abolishes the H₂S-induced inhibition of NLRP3, Caspase-1, and GSDMD expression in hypoxia-stimulated neurons. n = 3, * P < 0.05, ** P < 0.01. K , L Fluorescence staining and quantification of ROS in each group. Scale bar, 50 μm. n = 3, * P < 0.05, ** P < 0.01.

Article Snippet: Active Rac1 (GTP-bound) levels were quantified using the G-LISA® Activation Assay Kit (Cytoskeleton, CO, USA) following manufacturer guidelines.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Activity Assay, Immunoprecipitation, Purification, Inhibition, Fluorescence, Staining

Journal: Cell Death Discovery

Article Title: Hydrogen sulfide protects against spinal cord pyroptosis via persulfidation of Rac1 after lumbosacral plexus nerve injury

doi: 10.1038/s41420-025-02736-x

Figure Lengend Snippet: A , B Structural prediction of Rac1 and lists potential sulfhydrylation sites with modification scores. C Molecular docking model identifying Cys178 as the most favorable persulfidation site. D Western blot and quantification of Rac1 persulfidation (S-Rac1) in wild-type (WT) and C178S mutant neurons. n = 3, * P < 0.05, ** P < 0.01. E Kinetic simulation (50 ns) of Rac1 with H 2 S. F Analysis of radius of gyration and solvent accessible surface area, indicating structural activity alterations. G Western blot showing that H₂S fails to downregulate NLRP3, GSDMD, and Caspase-1 in C178S mutant neurons under hypoxia. n = 3, * P < 0.05, ** P < 0.01. H – J Immunofluorescence and quantification of NLRP3 with NeuN in neurons of each group. Scale bar, 50 μm. n = 3, * P < 0.05, ** P < 0.01.

Article Snippet: Active Rac1 (GTP-bound) levels were quantified using the G-LISA® Activation Assay Kit (Cytoskeleton, CO, USA) following manufacturer guidelines.

Techniques: Structural Proteomics, Modification, Western Blot, Mutagenesis, Solvent, Activity Assay, Immunofluorescence