active rho detection kit  (Cell Signaling Technology Inc)

Journal: The American Journal of Pathology

Article Title: Inhibiting the Secreted RGDKGE Collagen Peptide Selectively Controls CD8 + T-Cell Migration on Denatured Collagen-IV and Enhances Their Accumulation in Tumors

doi: 10.1016/j.ajpath.2025.09.008

Figure Lengend Snippet: RHOA and MYO9B play roles in the ability of the RGDKGE collagen peptide (P2) to modulate CD8 + T-cell migration on denatured collagen-IV (Den-Coll-IV). A and B: Whole cell lysates were prepared from Jurkat T cells treated with the P2 or control peptide (CP; A ) or dimethyl sulfoxide (DMSO) buffer control or RHOA inhibitor rhosin ( B ) and analyzed by Western blot analysis for total and activated RHOA using an active RHO detection kit. C and D: Quantification of Jurkat T-cell migration on denatured collagen-IV ( C ) or native collagen-IV (Nat-Coll-IV; D ) in the presence of DMSO or RHOA inhibitor rhosin. C: Data represent change in cell migration from four independent experiments with control DMSO treatment set to 100% for comparison. D: Data represent change in cell migration from three independent experiments with control DMSO treatment set to 100% for comparison. E and F: Cell lysates were prepared from Jurkat T cells treated with the P2 or CP ( E ) or DMSO or SRC non-receptor tyrosine kinase (SRC) inhibitor ( F ), and analyzed by Western blot analysis for MYO9B. G: Western blot analysis for MYO9B levels in control or MYO9B-specific siRNA-transfected Jurkat T cells. H and I: Quantification of control and MYO9B-specific knockdown Jurkat T-cell migration on denatured collagen-IV ( H ) or native collagen-IV ( I ). H: Data represent change in cell migration from four independent experiments with control siRNA treatment set to 100% for comparison. I: Data represent change in cell migration from two independent experiments with control siRNA treatment set to 100% for comparison. J: Quantification of MYO9B knockdown Jurkat T-cell migration on denatured collagen-IV in the presence of CP or P2. Data represent change in cell migration from three independent experiments with control CP treatment set to 100% for comparison. K: Example of F-actin polarization ( arrows ) in control or MYO9B-specific siRNA-transfected Jurkat T cells seeded onto denatured collagen-IV. L: Quantification of the mean percentage of F-actin polarized cells per ×400 field in control and MYO9B knockdown Jurkat T cells seeded onto denatured collagen-IV. Data represent percentage of F-actin polarized T cells from two independent experiments calculated from 10 ×400 microscopic fields per condition. Data are given as means ± SEM ( C , D , H – J , and L ). ∗ P < 0.05, ∗∗ P < 0.01. Scale bars = 20 μm ( K ).

Article Snippet: To detect active RHOA, the (Rhotekin-RBD) active RHO detection kit (Cell Signaling) was used.

Techniques: Migration, Control, Western Blot, Comparison, Transfection, Knockdown

Journal: Advanced Science

Article Title: Integrin α8‐Mediated Pericyte Morphogenesis Controls Blood‐Brain Barrier Integrity

doi: 10.1002/advs.202415374

Figure Lengend Snippet: Impact of ITGA8 Deficiency on Pericyte Morphology and Interactions with Endothelial Cells. A) Brain sections of ITGA8 iPCKO; H11‐GFP‐tdTomato mice and control mice at 40 days, immunostained for vessels. Scale bar: 40 µm. Higher magnification images in the right panels indicated by yellow boxes, Scale bar: 20 µm. B–D) Quantification of B) pericyte projection length, C) coverage, and D) number in brain sections at 40 days ( n = 6 mice, p = < 0.0001, 0.0001, 0.8753). E) Representative images of CD13+ pericyte coverage on CD31+ vessels in brain sections of ITGA8 iPCKO mice and control mice at 40 days. Scale bar: 40 µm. Higher magnification in right panels with yellow boxes, Scale bar: 20 µm. F) Transmission electron microscopy of brain microvessels in ITGA8 iPCKO and ITGA8 fl/fl mice at 40 days. EC: endothelial cell; PC: pericyte; L: lumen. The arrowhead indicates contracted pericyte bodies and diminished pericyte coverage on microvasculature, with pericytes appearing reduced interactions with neighboring endothelial cells in ITGA8 iPCKO mice. G) Double immunostaining for CD31 and N‐cadherin in brain sections at 40 days. Scale bar: 25 µm. Quantitative analysis of N‐cadherin relative MFI on CD31+ vessels in brain sections at 40 days ( n = 6 mice, p = 0.0018). H) GTP‐loaded RhoA levels measurement using a RhoA activation assay kit, which employs the pull‐down method to quantify active RhoA levels in HBPCs following hITGA8 and its knockdown ( n = 3 independent experiments). I) Quantification of RhoA activation status in HBPCs under ITGA8 overexpression versus knockdown conditions was performed using the G‐LISA RhoA activation assay ( n = 3 independent experiments, p = <0.0001, 0.0470). J) Images of ITGA8 manipulated HBPCs show that ITGA8 knockdown in HBPCs results in altered cell shapes and reduced projections compared to control cells, while ITGA8 overexpression enhanced cellular extensions. Additionally, the application of the ROCK inhibitor Y27632 to ITGA8 ‐overexpressing HBPCs reveals a reduction in the ITGA8 ‐induced morphological changes ( n = 3 independent experiments, all p < 0.0001). K) Western blot analysis of p‐MLC/MLC levels in the pericytes of ITGA8 iPCKO and ITGA8 fl/fl mice at 40 days ( n = 4 mice, p = 0.0003). L) Western Blot Analysis of p‐MLC in HBPCs depicting the levels of p‐MLC/MLC in HBPCs following ITGA8 knockdown or overexpression or treated with RhoA inhibitor Y27632 ( n = 3 independent experiments, all p < 0.0001). M) Confocal images of HBPC‐HBEC mixed cultures in fibrin gel on day 3, showing cellular interactions. HBPCs were labeled with a red CellTracker dye and HBECs with a green one ( n = 3 independent experiments, p = 0.0012). N) Confocal image showing interactions between GFP‐expressing HBPCs (green) and unlabeled HBECs following a 16 h co‐culture in Matrigel. Arrowheads point to altered cellular projections in pericytes. Arrowheads denote pericyte processes and intercellular networks, while arrows highlight endothelial‐aligned pericyte somata ( n = 3 independent experiments, p = 0.0009). Data represent mean ± SEM. Significance notations: ns ( p > 0.05), ** p < 0.01, **** p < 0.0001; Unpaired, 2‐tailed Student t‐ test was used to compare groups in (B–D), (G), (K), and (N). Comparisons between multiple groups were made using one‐way ANOVA test followed by Tukey's HSD post hoc test in (I–J) and (L).

Article Snippet: GTP‐loaded RhoA levels were determined using a RhoA activation assay kit (CST, 8820) based on a pull‐down method.

Techniques: Control, Transmission Assay, Electron Microscopy, Double Immunostaining, Activation Assay, Knockdown, Over Expression, Western Blot, Labeling, Expressing, Co-Culture Assay

Journal: Life science alliance

Article Title: The Ragulator complex and lysosomal calcium release are crucial for cell migration.

doi: 10.26508/lsa.202403015

Figure Lengend Snippet: Figure 4. Ouabain ameliorates inflammation by inhibiting cell migration. (A) Effect of ouabain on migration of WT-THP1 cells. WT-THP1 cells were preincubated with ouabain (50 or 100 nM) or DMSO for 2 h. Chemotaxis was induced in response to CCL2 (25 ng/ml). N = 5. (B) Effects of ouabain on MLC phosphorylation. WT-THP1 cells treated with ouabain (50 or 100 nM) or DMSO for 6 h were lysed, and Western blotting was performed using an anti-PMLC antibody. N = 5. (C) Detection of active Rac1 and RhoA. Rac1 expression was assessed using Active Rac1 Detection Kit. Representative Western blots are shown, using the anti-Rac1 antibodies at left. N = 5. Active RhoA levels were assessed using Active RhoA Detection Kit. Representative

Article Snippet: Then, the active form of RhoA was pulled down with a GST-Rhotekin-RBD fusion protein and immunoprecipitated with glutathione resin (Active RhoA Detection Kit; Cell Signaling Technologies).

Techniques: Migration, Chemotaxis Assay, Phospho-proteomics, Western Blot, Expressing