cdc42 pulldown activation assay kits  (Cytoskeleton Inc)

Journal: BMC Biology

Article Title: The cytoskeleton adaptor protein Sorbs1 controls the development of lymphatic and venous vessels in zebrafish

doi: 10.1186/s12915-024-01850-z

Figure Lengend Snippet: Sorbs1 controls EC adhesive properties via RhoGTPases in vitro and in vivo. A Cdc42, Rac1, and RhoA activity in HUVECs transfected with control (Ctl) or Sorbs1 siRNA. Histogram is from Western blot densitometric analysis of three independent pull-down experiments and represents the ratio between bound active- and total amount of each RhoGTPase in the lysate, relative to control cells (*** P < 0.001 , * P < 0.05 , ns = non-significant, Student’s t -test). B Confocal pictures of peripheral F-Actin (phalloidin staining) in Ctl or Sorbs1 siRNA transfected HUVECS treated ( +) or not ( −) with the C3 RhoA inhibitor. Images are shown using an intensity-based look-up table (from blue = low to red = high). Numbers represent the average signal intensity ± SD in each condition. Scale bar represents 50 μM. C Adhesion complexes were analyzed by confocal microscopy after immunostaining of Paxillin and phospho-Paxillin (p-Paxilin) in HUVECs transfected with control or Sorbs1 siRNA. Scale bar represents 20 μm. Nascent adhesions (NA) and focal complexes (Fx) are characterized by their small size, peripheral location and high p-Paxillin/Paxillin ratio content (arrowheads). Larger and more mature focal adhesion (FA) were defined as bigger than 1 μm 2 , and their proportion in each condition was quantified ( n = 21; * P < 0.05 , Student’s t -test). D Representative micrographs and quantification of adhesion assays performed with HUVECs transfected with control or Sorbs1 siRNA as described in the “ ” section. Scale bars represent 100 μm ( n = 3 independent experiments; ** P < 0.01 , Student’s t -test). E Representative live confocal images (Z-maximum intensity projection) of emerging filopodia in the CVP of 26 hpf Tg(LIFEACT:mKate2) embryos injected with Ctl or Sorbs1 morpholino at 5 ng/ul. Enrichment of actin at the cell cortex from the boxed area is visualized using a color look-up table (intensity scale). The number of filopodia per 40 μm and the cortical enrichment of the F-actin signal is indicated for both conditions in the table. Scale bar represents 40 μm ( n = 7/WT and n = 9/Sorb1 Mo, * P < 0.05 , Mann–Whitney U -test). F WT and sorbs1 −/− embryos were treated or not with RhoA inhibitor at 26 hpf, and the percentage of aISV/vISV at 48 hpf was quantified ( n = number of embryos; * P < 0.05 ; ns = non-significant; χ 2 with Yates correction). G Quantification of PL number in 10 somites at 54 hpf in WT and sorbs1 −/ . − embryos injected with RhoA inhibitor at 26 hpf or left untreated ( n = number of embryos; * P < 0.05 ; ns = non-significant; Mann–Whitney U -test)

Article Snippet: The levels of active Rac1 and Cdc42 were measured using the Rac1 and CDC42 Activity Assay (Cytoskeleton Inc., BK035 and BK034, respectively); cells were lysed in a buffer containing 5 mM DTT, 50 mM Tris pH 7.2, 1% tritonx-100 (10%), 0.5% deoxycholate (20%), 0.1% SDS (20%), and 500 mM NaCl 5 M. Extracts were then incubated 1 h at 4 °C with GST-PAK beads.

Techniques: Adhesive, In Vitro, In Vivo, Activity Assay, Transfection, Western Blot, Staining, Confocal Microscopy, Immunostaining, Injection, MANN-WHITNEY

Journal: Advanced Science

Article Title: A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42‐mediated GSK‐3β/β‐catenin Signaling

doi: 10.1002/advs.202307850

Figure Lengend Snippet: DA directly bound to Cdc42 and inhibited its activity. A) Heatmap indicating ‐log 2 (FC) of Δ T m at each temperature for Uba5, Igf2bp1, Cdc42, Dctn1 and Plod2. Results are shown from 2 replicate experiments. B) Effects of the knockdown of Cdc42, Uba5, and Dctn1 on the protein levels of α‐SMA measured by western blotting ( n = 3 per group). C) The HPCL/MS/MS analysis of NRK‐49F treated with vehicle or DA . D) CETSA analysis of intracellular binding between DA and Cdc42. Protein levels were investigated at different temperatures under the treatment of DA (5 µM) in NRK‐49F cells for 1 h. The graph shows the quantification of Cdc42 protein versus temperature points based on Western analyses ( n = 3 per group). E) Surface plasmon resonance (SPR) analysis of interactions between DA or ZCL278 and Cdc42. F) Immunoblots of Cdc42 in NRK‐49F cells treated with vehicle or DA ( n = 3 per group). G) Analysis of the active and total Cdc42 in NRK‐49F cells treated with vehicle or DA by pull‐down assay. Quantitative analysis of the grey value for active Cdc42/total Cdc42 ratio using ImageJ software ( n = 3 per group). H) Representative immunofluorescence microscopy of active Cdc42. Serum‐starved NRK‐49F cells were treated with vehicle, 5 µM DA , or 5 µM ZCL278. Cells were probed with an active Cdc42 antibody (green). Nuclei were visualized by DAPI (blue). Scale bar, 20 µm. I) Analysis of the active and total Cdc42 in kidneys from mice treated with vehicle, low‐dose, or high‐dose DA by pull‐down assay ( n = 6 per group). Results were quantified by ImageJ software. J) Molecular docking of Cdc42 and DA . K) CETSA analysis of intracellular binding between DA and wild‐type or mutant Cdc42 ( n = 3 per group). Data were analyzed by two‐way ANOVA followed by Bonferroni's multiple comparisons test. Control, CTL; vehicle, Veh; WT, wild‐type; F, Phe; K, Lys; L, Leu. Data are presented as means ± SEM (B, D, F, G, I, and K) and were analyzed using one‐way ANOVA followed by a Bonferroni's multiple comparisons test unless otherwise stated. ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared with CTL or sham group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with Veh group or between groups indicated by a line; ns, not significant.

Article Snippet: The assay was performed according to the instructions of Cdc42 activation assay biochem kit (BK034, Cytoskeleton) according to the instructions.

Techniques: Activity Assay, Western Blot, Tandem Mass Spectroscopy, Binding Assay, SPR Assay, Pull Down Assay, Software, Immunofluorescence, Microscopy, Mutagenesis

Journal: Advanced Science

Article Title: A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42‐mediated GSK‐3β/β‐catenin Signaling

doi: 10.1002/advs.202307850

Figure Lengend Snippet: DA activated p‐PCKζ/p‐GSK‐3β‐mediated β‐catenin phosphorylation at S33/37/T41 by targeting Cdc42. A, B) Immunoblots showing the inhibitory effects of DA and ZCL278 on fibrotic markers in A) TGF‐β1‐activated NRK‐49F cells ( n = 3 per group) and B) UUO mice ( n = 6 per group). C, E) Influence of Cdc42 knockdown on (C) fibrosis‐related proteins and (E) p‐PKCζ/p‐GSK‐3β/p‐β‐catenin protein levels in NRK‐49F cells ( n = 3 per group). NRK‐49F cells were transfected with si‐Cdc42 or negative control siRNA (si‐nc) and incubated with TGF‐β1+Veh or TGF‐β1+ DA for C) 48 h or E) 12 h. D, F) Immunoblot analysis of D) fibrotic indicators and F) p‐PKCζ/p‐GSK‐3β/p‐β‐catenin axis in NRK‐49F cells transfected with constitutively active Cdc42 plasmid (Cdc42 Q61L ) ( n = 3 per group). The post‐transfected NRK‐49F were treated with TGF‐β1+Veh or TGF‐β1+ DA for D) 48 h or F) 12 h. G) Schematic diagram summarizing how active Cdc42 modulates p‐PKCζ/p‐GSK‐3β/p‐β‐catenin axis. H) Immunoblot analysis of p‐PKCζ and p‐GSK‐3β in mice kidneys of each group on the seventh days after sham or UUO surgery ( n = 6 per group). Control, CTL; vehicle, Veh; NC, negative control; Ser, S; Thr, T. Data are presented as means ± SEM (A, B, C, D, E, F, and H) and were analyzed using one‐way ANOVA followed by a Bonferroni's multiple comparisons test. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 compared with CTL group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with Veh group or between groups indicated by a line; ns, not significant.

Article Snippet: The assay was performed according to the instructions of Cdc42 activation assay biochem kit (BK034, Cytoskeleton) according to the instructions.

Techniques: Western Blot, Transfection, Negative Control, Incubation, Plasmid Preparation

Journal: Advanced Science

Article Title: A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42‐mediated GSK‐3β/β‐catenin Signaling

doi: 10.1002/advs.202307850

Figure Lengend Snippet: Higher expressions of Cdc42 were seen in both UUO mice and CKD patients. A) Immunoblot analysis of Cdc42 in mice kidneys of each group on the seventh day after sham or UUO surgery ( n = 3 per group). B) Cdc42 transcript expression analysis for human kidneys from healthy controls (HC) and CKD patients in two public RNA‐sequencing datasets, GSE66494 and GSE115857. C) Correlation of the mRNA levels between Cdc42 and α‐SMA ( Acta2 ), collagen I ( Col1a1 ), collagen III ( Col3a1) , and fibronectin ( Fn1 ) were measured by Pearson's correlation coefficient (r) in GSE115857. The data is shown from 7 HC and 23 CKD patients. D) Immunohistochemical staining of Cdc42 in kidney sections from HC ( n = 5) and CKD ( n = 12). Scale bar = 200 µm. Blue dashed lines outline the glomerulus and red dashed lines for tubule. Data are presented as means ± SEM (A, B, and D) and were analyzed by students’ t‐test. ### p < 0.001, #### p < 0.0001 compared with sham or HC group.

Article Snippet: The assay was performed according to the instructions of Cdc42 activation assay biochem kit (BK034, Cytoskeleton) according to the instructions.

Techniques: Western Blot, Expressing, RNA Sequencing Assay, Immunohistochemical staining, Staining