rac1 glisa activation assay  (Cytoskeleton Inc)

Journal: Scientific Reports

Article Title: Plakoglobin does not participate in endothelial barrier stabilization mediated by cAMP

doi: 10.1038/s41598-025-93756-1

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: To determine the intracellular concentration of active, GTP-bound Rac1 and RhoA proteins, a colorimetric-based GLISAs for Rac1 (Cytoskeleton, #BK128) and RhoA (Cytoskeleton, #BK124) were used.

Techniques: Transduction

Journal: Scientific Reports

Article Title: Plakoglobin does not participate in endothelial barrier stabilization mediated by cAMP

doi: 10.1038/s41598-025-93756-1

Figure Lengend Snippet: Effect of PG deficiency on central structural and signalling molecules, cAMP levels and small Rho GTPases activity. ( a ) Representative Western blot, N ≥ 5 and ( b ) PCR, N ≥ 3 analyses of junctional and junctional-related molecules revealing the protein and mRNA expression levels, respectively, in WT and PG-KO cells. α-tubulin was used as loading control for WB. Equal loading for the PCR analyses was validated by B2M expression. ( c ) Bar diagram depicting the relative protein expression of each analysed target of interest in PG-KO when compared to WT cells. ( d ) Bar diagram presenting the mRNA expression in PG-KO, relative to WT cells. ( e ) Western blot analysis illustrating the relative expression of signaling molecules such as Epac1, Rac1 and RhoA upon PG depletion with respective α-tubulin loading control; N ≥ 6 ( f ) Bar diagram representing the protein expression of the targets tested in ( e ), relative to WT, after densitometric measurements of the signal intensity were accomplished. ( g ) Bar graph illustrating the activity of Rac1 and RhoA GTPases measured by G-LISA; N ≥ 7 h) Levels of intracellular cAMP in untreated WT and PG-KO, detected by ELISA; N = 6. Data are presented as mean ± SEM; the following values were considered significant * p ≤ 0,05; ** p ≤ 0,01; **** p ≤ 0,0001.

Article Snippet: To determine the intracellular concentration of active, GTP-bound Rac1 and RhoA proteins, a colorimetric-based GLISAs for Rac1 (Cytoskeleton, #BK128) and RhoA (Cytoskeleton, #BK124) were used.

Techniques: Activity Assay, Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Plakoglobin does not participate in endothelial barrier stabilization mediated by cAMP

doi: 10.1038/s41598-025-93756-1

Figure Lengend Snippet: Effect of F/R-mediated intracellular cAMP elevation on barrier integrity and activity of members of Rho family of small GTPases in WT and PG-depleted cells. ( a ) Analysis of barrier integrity of WT and PG-KO cells exposed to vehicle or F/R. The segmented orange line indicates the time of mediator application. “*” underlined with green or red line denotes the time window of significant difference in TEER between Vehicle and F/R-treated monolayers in WT or PG-KO, respectively, N = 8. ( b ) Bar diagram displaying the intracellular cAMP levels after 1 h of treatment, assessed by ELISA; N = 6 ( c ) Western blot analysis visualizing the relative protein expression of Rac1 and RhoA in WT and PG-KO cells treated either with vehicle or F/R. α-tubulin was used to charge equal loading. Non-contiguous RhoA bands originating from the same membrane are distinct by a vertical black line. An equal exposure time was used for detection of the specific bands. Original blots are presented in Figure S6 d, provided in the Supplementary info file; N = 3 ( d ) Bar graph representing the respective densitometric measurement of the bands enclosed in ( c ). Within the respective cell line, the expression was reported as relative to Vehicle; to validate effective treatment with F/R, the phosphorylation status of VASP was assessed by building a ratio between phosphorylated and total VASP (sum of phosphorylated- and not phosphorylated- VASP). The data are presented as relative to the respective Vehicle; N ≥ 10 ( e ) Modulation of Rac1 and RhoA GTPase activities due to F/R treatment in WT and PG-KO cells, assessed by G-LISA; N ≥ 3. Data are presented as mean ± SEM; * p ≤ 0,05; ** p ≤ 0,01**** p ≤ 0,0001.

Article Snippet: To determine the intracellular concentration of active, GTP-bound Rac1 and RhoA proteins, a colorimetric-based GLISAs for Rac1 (Cytoskeleton, #BK128) and RhoA (Cytoskeleton, #BK124) were used.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Membrane, Phospho-proteomics

Journal: bioRxiv

Article Title: Volumetric Compression Shifts Rho GTPase Balance and Induces Mechanobiological Cell State Transition

doi: 10.1101/2023.10.08.561452

Figure Lengend Snippet: (A) Kymographs showing 120-min cell edge ruffling after 24-hour cell attachment in compression media. Scale bar, 25 μm. (B) Profiling cell boundaries for 120 min (2 min interval) and measuring area fluctuation of individual cells, defined by the standard deviation of the mean area of the cell over time (also see Extended Data Fig.1A, Supplementary Video 2). N=15 cells per condition from three independent experiments. Scale bar, 50 μm. (C) Fluorescent images of VASP and F-actin of the cells under varied compression. The close-up images show the distribution of VASP with respect to the actin. The fluorescent images were used to characterize: (D) the ratio of lamellopodia in the whole cell area and the absolute area of the lamellopodia. N=40, 40, and 52 cells respectively from three independent experiments. (E) High-resolution confocal images show the relative distribution of p-MLC on the actin cytoskeleton. (F) Fluorescent images of Myosin-X and F-actin of the cells under varied compression. Scale bar, 10 μm. (G) Fluorescent images of Vinculin and F-actin of the cells under varied compression. The close-up images show the distribution of vinculin with respect to the actin. (H) Vinculin puncta were used to determine the size distribution of focal adhesions and the total adhesion area of individual cells for varied conditions. N=21, 20, and 22 cells respectively from two independent experiments. FA measurements from all cells were pooled to plot the size distribution. (I) Quantifications (GLISA) of active (GTP-bound) RhoA and Rac1 of the cells cultured under varied compression for 24 hours compared to the cells culture in the isotonic condition. Mean±s.e.m. Statistic differences were calculated by one-way ANOVA with a Dunnett’s multiple comparisons test. (J) Proposed model of volumetric compression regulating Rho/Rac balance. For (C,E,F), scale bars in the close-up images, 20 μm; scale bars in the close-up images, 10 μm. Statistic analyses in (B,D,G) were calculated by one-way ANOVA with a Tukey multiple comparisons test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns: not significant).

Article Snippet: Colorimetric-based GLISA assays (Cytoskeleton, Inc.) were used to quantify RhoA and Rac1 activity.

Techniques: Cell Attachment Assay, Standard Deviation, Cell Culture

Journal: bioRxiv

Article Title: Volumetric Compression Shifts Rho GTPase Balance and Induces Mechanobiological Cell State Transition

doi: 10.1101/2023.10.08.561452

Figure Lengend Snippet: GLISA assays were used to determine levels of Rac1 and RhoA activities after 6-hour treatment of GTPases activator CN04 (GTPases act.). This activator shows a long-lasting effect on Rac1 activation in 4% PEG-compressed SNU475 cells but not on the RhoA. Plots are shown as Mean±s.e.m. n=9 replicates for Control, and 7 replicates for GTPase activator, from three independent experiments. Two-tailed t-tests are used to determine the statistical difference (****p<0.0001; ns: not significant).

Article Snippet: Colorimetric-based GLISA assays (Cytoskeleton, Inc.) were used to quantify RhoA and Rac1 activity.

Techniques: Activation Assay, Control, Two Tailed Test