rac1 cdc42 activation assay combo kit  (Cell Biolabs Inc)

Journal: bioRxiv

Article Title: Astrocyte targeted SMN1 gene therapy and forskolin application improves astrocyte filopodia actin defects and motor neuron synaptic dysfunction in human SMA disease pathology

doi: 10.64898/2026.03.26.714618

Figure Lengend Snippet: (A) Representative images from FiloQuant analysis used to quantify filopodia density in healthy and SMA patient astrocyte monocultures at baseline and after stimulating actin remodeling using the ATP depletion and recovery assay (NaN3 +R). CMFDA dye was used to visualize the cells (grey signal) and filopodia quantified by FiloQuant tool are highlighted in magenta. Scale bar: 30µm. (B) Imaris surface and mask tool applied on to individual healthy and SMA patient astrocyte cells at baseline and after ATP depletion and recovery assay. Scale bar: 15µm. (C) Quantification of filopodia density from FiloQuant analysis across baseline and treatment conditions for healthy and SMA patient astrocyte samples. One-way ANOVA with Bonferroni multiple comparison statistical testing; ****p<0.0001. (D) Optic density readout from CDC42-GTP G-LISA assay measuring the activated form of CDC42 across baseline and treatment conditions for healthy and SMA patient astrocyte samples. One-way ANOVA with Bonferroni multiple comparison statistical testing, p-values not statistically significant. N=5 (biological replicates), n= 4 (technical replicates).

Article Snippet: The colorimetric CDC42 G-LISA GTPase activation assay kit (Cytoskeleton, Inc) was utilized to assess the amount of active CDC42-GTP within healthy and SMA astrocyte cultures at baseline and after actin stimulation via the ATP depletion and recovery assay.

Techniques: Comparison

Journal: bioRxiv

Article Title: Astrocyte targeted SMN1 gene therapy and forskolin application improves astrocyte filopodia actin defects and motor neuron synaptic dysfunction in human SMA disease pathology

doi: 10.64898/2026.03.26.714618

Figure Lengend Snippet: (A) Schematic depicting downstream cellular mechanism activated by forskolin treatment. Forskolin application activates adenylyl cyclase (AC) at the plasma membrane, leading to an increase in intracellular levels of cyclic adenosine mono-phosphate (cAMP) and downstream activation of the protein kinase A (PKA) pathway. This can modulate SMN protein levels via phosphorylation and stabilization, in addition to actin remodeling via activation of CDC42. (B) Representative confocal images of SMN signal (grey) in SMN re-expressing SMA astrocytes at baseline (SMN:GFP UTX) and after 10µM forskolin treatment (1hr 30min incubation; SMN:GFP (F). VIMENTIN signal (magenta) was used to visualize astrocytes Scale bars: 30µm. (C) Quantification of SMN puncta in SMA patient astrocytes and SMN re-expressing SMA astrocytes and after 10µM forskolin treatment. Unpaired t-test; ns=not significant. Imaris surfaces outlining cell morphology (D) and quantification of filopodia density (E) for healthy and SMA astrocytes after forskolin treatment and forskolin treatment added during recovery period of actin remodeling (NaN3 + R(F). Imaris surfaces outlining cell morphology and quantification of filopodia density for SMN:FLAG re-expressing SMA astrocytes (F&G) and SMN:GFP re-expressing SMA astrocytes (H&I) . Conditions include baseline (UTX), forskolin treatment (F), actin remodeling via ATP depletion and recovery assay (NaN3 + R) and forskolin treatment added during recovery period of actin remodeling (NaN3 + R(F). Average filopodia density of healthy and SMA astrocyte datasets (featured in ) is depicted as threshold dotted lines in graphs. One-way ANOVA with Bonferroni multiple comparison statistical testing; **p=0.0082, ***p=0.0005. N=5 (biological replicates), n= 3 (technical replicates)

Article Snippet: The colorimetric CDC42 G-LISA GTPase activation assay kit (Cytoskeleton, Inc) was utilized to assess the amount of active CDC42-GTP within healthy and SMA astrocyte cultures at baseline and after actin stimulation via the ATP depletion and recovery assay.

Techniques: Clinical Proteomics, Membrane, Activation Assay, Phospho-proteomics, Expressing, Incubation, Comparison

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: Natural killer cells from old adults show specific changes in the transcriptome and regulation of distinct signaling pathways. (A) Heatmap representing the global gene expression pattern in NK cells isolated from young and old healthy human donors. The color key represents the expression intensity from blue color (low expression) to red color (high expression), while white color represents no expression (0 value). (B) Pathway analyses of transcriptome data of NK cells isolated from young and old donors, based on upregulated and downregulated gene sets. The activated (upper graph) and inhibited (lower graph) pathways in NK cells isolated from old donors in comparison to young donors are presented. (C, D) Pull down western blot analysis of total and active Cdc42 of lysates from NK cells of donor set 1 (C) and donor set 2 (D), each donor set consisting of NK cells from 6 pooled young donors, 6 pooled old donors and 6 pooled CASIN (5 μM) treated NK cells from old donors. (E) Graph showing densitometric analysis of active Cdc42 level in the vehicle treated NK cells from young, old donors and CASIN treated NK cells from old donors. The densitometric value of NK cells from young donor set 1 was set to one fold (reference). The relative densitometric values from other groups relative to reference was then computed and plotted. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance. (F, G) Representative immunofluorescence staining and distribution of Cdc42 (green) and tubulin (red) in NK cells from young and old donors treated with vehicle, and NK cells from old donors treated with CASIN. Immunostaining was performed on NK cells of 3 different donors per experimental group. (H, I) Representative immunofluorescence micrograph for the quantification of the spatial distribution of Cdc42 (G) and tubulin (H) of young vehicle treated NK cells (first panel), old vehicle treated NK cells (middle panel) and old CASIN treated NK cells (last panel). Fifteen single cells were used for each analysis. (J, K) Quantification of Cdc42 (H) and tubulin (I) polarity score of data shown in F and G, as mentioned in Section . Data were represented as mean ± SEM, N = 15 individual cells for each group. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance.

Article Snippet: The active cdc42 detection kit (Cell Signaling Technology Cat. #8819) was used in the cdc42 pull down assay and was done accordingly as stated in the manufacturer's protocol.

Techniques: Protein-Protein interactions, Gene Expression, Isolation, Expressing, Comparison, Western Blot, Immunofluorescence, Staining, Immunostaining

Journal: Aging Cell

Article Title: Overactivation of Cdc42 GTPase Impairs the Cytotoxic Function of NK Cells From Old Individuals Towards Senescent Fibroblasts

doi: 10.1111/acel.70398

Figure Lengend Snippet: CASIN treatment improves the cytotoxic ability of Natural killer cells from old humans and mice. (A) Graphical illustration of experimental plan, where young NK cells treated with vehicle and old NK cells treated with either vehicle or CASIN for 8 h and thereafter subjected to co‐culture with target senescent HDF exerting their differential killing ability. (B) Representative histograms depicting the killing ability of different experimental groups as measured by flow cytometry. Peak at the left side of histogram, showing the dead senescent HDF population with percentage of dead cells. (C) Quantification of the percentage of target senescent HDF death executed by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of senescent fibroblast death) ± SEM. N = 4. (D) Representative histograms show the distribution of K562 killing by young NK cells treated with vehicle, and old NK cells treated with either vehicle or CASIN. Peak at the left side of histogram, showing the dead K562 population with percentage of dead cells. (E) Graph shows the percentage of target cell (K562) death mediated either by young NK cells treated with vehicle or by old NK cells treated with either vehicle or CASIN. Data were represented as mean (Percentage of K562 lysis) ± SEM. N = 4. One‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups in C and E. (F) Illustration of the experimental design for treatment of young mice (average age 120 days) treated with vehicle and old mice (average age 650 days) treated with either vehicle or CASIN. Following treatment, NK cells were isolated from spleen and bone marrow and subjected to co‐cultures with murine dermal fibroblasts (MDF) derived from old mice (average age 650 days). (G) Flow cytometry with representative histograms depicting old/senescent MDF killing by NK cells isolated from bone marrow (left panel) and spleen (right panel) of vehicle and CASIN treated old mice. Peak at the left side of histogram, showing the dead old MDF population with percentage of dead cells. (H) Quantification of the percentage of old/senescent MDF killing by NK cells isolated from bone marrow and spleen of young and old mice treated with vehicle and old mice treated with CASIN. Data were represented as mean (percentage of old/senescent MDF lysis) ± SEM, N = 4, where each group contains pool of NK cells isolated from 4 different mice of same treatment group. Two‐way ANOVA, followed by Bonferroni multiple comparison test was used to find the significance among the groups. (I) Graphical summary. Unrestrained Cdc42 activity causes failure of old NK cells to kill senescent fibroblasts. Unrestrained Cdc42 activity disrupts the microtubular network and impaired mitochondrial ATP resulting in reduced conjugation, and impaired degranulation of lytic vesicles into the synaptic cleft with reduced cytotoxicity. CASIN can attenuate all these steps and in part attenuate the killing of senescent fibroblasts (senescent HDF).

Article Snippet: The active cdc42 detection kit (Cell Signaling Technology Cat. #8819) was used in the cdc42 pull down assay and was done accordingly as stated in the manufacturer's protocol.

Techniques: Co-Culture Assay, Flow Cytometry, Lysis, Comparison, Isolation, Derivative Assay, Activity Assay, Conjugation Assay