Journal: Nature cell biology

Article Title: An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

doi: 10.1038/ncb3592

Figure Lengend Snippet: ( a,b ) FRET analysis of Cdc42 activity at the apical aspect of MDCK cells conditionally expressing Dbl3-myc. ( c,d ) Confocal microscopy of pMLC and F-actin at the apical membrane domain of MDCK cells conditionally expressing Dbl3-myc induced with tetracycline in the absence or presence of Cdc42 inhibitor. White arrowheads highlight the apical membrane cortex labelled for F-actin. ( e,f ) Analysis of aPKCζ localization in MDCK cells with tetracycline-inducible Dbl3-myc expression. White arrowheads point to the lateral junctional complex. ( g ) Schematic diagram illustrating activation of two effector mechanisms, MRCK/Myosin-II and Par6-aPKC by Dbl3 stimulated apical Cd42-activation. Quantifications show shown are (b) box blots (25th to 75th percentiles, with a line at the median; whiskers extend to the max/min data points; n=36 cells -Tet and n=46 cells +Tet; p value was calculated with a Wilcoxon test), or (d, f) based on n=3 independent experiments and showing the data points, means ± 1 SD (in black), the total number of cells analysed for each type of sample across all experiments, and p-values derived from t-tests. Scale bars: 10 μm.

Article Snippet: Blebbistatin (10μM final concentration), the Cdc42 inhibitor ML141 (10μM), and the ROCKI/II inhibitor GSK 269962 (10nM) were purchased from Tocris Bioscience.

Techniques: Activity Assay, Expressing, Confocal Microscopy, Membrane, Activation Assay, Derivative Assay

Journal: Nature cell biology

Article Title: An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

doi: 10.1038/ncb3592

Figure Lengend Snippet: ( a,b ) Levels of EGFP-Lifeact (EGFP-LA), Par polarity and brush border proteins localized at the apical membrane domain in MDCK cells conditionally expressing Dbl3-myc, treated with blebbistatin and followed by washout (WO). ( c,d ) Levels of EGFP-Lifeact (EGFP-LA) enrichment at the apical membrane domain of MDCK cells conditionally expressing Dbl3-myc following blebbistatin treatment and washout in the absence or presence of BDP5290 to inhibit catalytic activity of MRCK. ( e-i ) Measurements of co-localization coefficients of PAR polarity and brush border proteins at the apical membrane domain in MDCK cells conditionally expressing Dbl3-myc transiently inhibited with blebbistatin followed by wash out for 2 hours (green indicates co-localisation, see also and ; arrowheads point to apical membrane). ( j ) Schematic model of polarity induced by apical stimulation of Cdc42, activating a dual effector mechanism to generate asymmetric actomyosin contractions, apical PAR domain formation and membrane morphogenesis. Bright green represents co-localization of Par and brush border proteins. ( k-n ) Quantification of confocal sections of pupal retinas stained for the apical markers aPKC and Moesin comparing wild type cells and gek mutant cells ( k, l ), or wild type cells and cells mutant for gek expressing Sqh EE ( m, n ). Quantifications in panels b, d, f, h, and i are based on n=3 independent experiments and shown are the data points, means ± 1 SD (in black), the total number of cells analysed for each type of sample across all experiments, and p-values derived from t-tests. Quantifications in panels k-n of protein expression levels compare measurements from wild type and neighbouring mutant cells (paired within sections; see for representative images); k, n=10 animals; l, n=8 animals; m and n, n=7 animals; p values were calculated with t-tests). Scale bars: 10 μm.

Article Snippet: Blebbistatin (10μM final concentration), the Cdc42 inhibitor ML141 (10μM), and the ROCKI/II inhibitor GSK 269962 (10nM) were purchased from Tocris Bioscience.

Techniques: Membrane, Expressing, Activity Assay, Staining, Mutagenesis, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: Endothelial cells-derived SEMA3G suppresses glioblastoma stem cells by inducing c-Myc degradation

doi: 10.1038/s41418-025-01534-3

Figure Lengend Snippet: A The ubiquitination level of c-Myc in GSC07 cells treated with or without ML141 (10 μmol/l) for 24 h. B , C c-Myc level in T98G cells transfected with GFP-Tagged Cdc42 WT , Cdc42 T17N or Cdc42 Q61L . D Schematic diagram depicting the plasmid construction for bimolecular fluorescence complementation assays. E Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between Cdc42 and WWP2 . F HEK-293 cells were transfected with CrN173-tagged Cdc42 T17N or Cdc42 Q61L , and VC155-tagged WWP2 for 48 h. CFP signal that represents Cdc42 binding to WWP2 were determined using confocal. Scale bar, 5 μm. G HEK-293 cells were transfected with Flag-tagged c-Myc, HA-tagged ubiquitin, MYC-tagged WWP2 and GFP-tagged Cdc42 T17N or Cdc42 Q61L for 48 h. The ubiquitin level of c-Myc and the binding of Cdc42 to WWP2 were determined by co-immunoprecipitation. H Schematic illustration of the bimolecular fluorescence complementation assay used to assess the interaction between WWP2 and Cdc42 or c-Myc. I HEK-293 cells were incubated with or without SEMA3G (200 ng/ml) for 48 h after co-transfected with CrN173-tagged Cdc42 WT or VN173-tagged c-Myc and VC155-tagged WWP2 for 12 h. CFP signal that represents Cdc42 binding to WWP2 and Venus signal that represents WWP2 binding to c-Myc were captured using confocal. Scale bar, 5 μm. J HEK-293 cells that were co-transfected with GFP-tagged Cdc42 WT , MYC-tagged WWP2, Flag-tagged c-Myc, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The binding of Cdc42 WT to WWP2 and the ubiquitination level of c-Myc were determined by co-immunoprecipitation. K HEK-293 cells were co-transfected with GFP-tagged Cdc42 Q61L and MYC-tagged WWP2, Flag-tagged c-MYC, and HA-tagged ubiquitin (UB) for 12 h, followed by the treatment with or without SEMA3G (200 ng/ml) for 48 h. The protein level of c-Myc and ubiquitination level of c-Myc were determined by co-immunoprecipitation. L Schematic diagram illustrating the potential mechanism by which Cdc42 regulates WWP2 binding to c-Myc and promotes c-Myc degradation. All the western blot bands represent one of the three independent experiments.

Article Snippet: USA), or ML141 (MedChemExpress, NJ, USA) according to the experimental design.

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Fluorescence, Bimolecular Fluorescence Complementation Assay, Binding Assay, Immunoprecipitation, Incubation, Western Blot

Journal: Experimental & molecular medicine

Article Title: SAMHD1-induced endosomal FAK signaling promotes human renal clear cell carcinoma metastasis by activating Rac1-mediated lamellipodia protrusion.

doi: 10.1038/s12276-023-00961-x

Figure Lengend Snippet: Fig. 5 SAMHD1 regulates Rac1 activity and enhances ccRCC cell migration. a GO analysis was performed, and subcategories in the molecular function category were highly correlated with the high expression of SAMHD1. b, c Subcategories of the molecular function category related to cell migration. **p < 0.01. d A G-LISA-based assay was performed to measure GTP-bound Rac1 activity in SAMHD1-knockdown SN12C cells and SAMHD1-overexpressing Caki-1 cells. Data are presented as the mean ± SD. The data shown are representative of three independent experiments. **p < 0.01. e GTP-bound-cdc42 activity in SAMHD1-knockdown SN12C cells and SAMHD1-overexpressed Caki-1 cells. Data are presented as the mean ± SD. The experiments were independently performed three times. *p < 0.05; ***p < 0.001. f Wound-healing assay of cell migration ability with FAK inhibitor (PF573228), Rho A inhibitor (fasudil), Rac1 inhibitor (NSC23766), and cdc42 inhibitor (ML141). SAMHD1- knockdown SN12C cells were treated with each corresponding inhibitor at 10 μM over 24 h. The relative wound-healing area was normalized to that of the controls. Data are presented as the mean ± SD. The data shown are representative of three independent experiments. ***p < 0.001. g A wound healing assay was performed as in (f) in SAMHD1-overexpressing Caki-1 cells. The relative wound-healing area was normalized to that of DMSO-treated controls. *p < 0.05; ***p < 0.001. Data are presented as the mean ± SD of three independent experiments.

Article Snippet: NSC23766 (#S8301), ML141 (#S7686), and Dynasore (#S8047) were obtained from Selleckchem (Houston, TX, USA).

Techniques: Activity Assay, Migration, Expressing, Knockdown, Wound Healing Assay

Journal: Nature Communications

Article Title: CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection

doi: 10.1038/s41467-023-36398-z

Figure Lengend Snippet: a Schematic of macropinocytosis and the Clathrin Independent Carrier (CLIC) pathways, their effectors and inhibitors. b GII.4 replication in the presence of EIPA (Na + /H + exchanger inhibitor), ML141 (Cdc42 inhibitor), Wiskostatin (N-WASP inhibitor), NSC23766 (RAC1 inhibitor), CT04 (RhoI inhibitor), Blebbistatin (myosin inhibitor), and LY29402 (PI3K inhibitor) at 1 h (gray) and 24 h (orange). c GII.4 replication in the presence of Arf1 inhibitor Golgicide A (GCA at 1 h (black) and 24 h (blue). d GII.4-induced tubular carriers at 1 h (37 o C) detected using guinea pig anti-Sydney VLP polyclonal antibody (Gp Syd-pAb) for viral capsid (green) and phalloidin for actin (red). Images were taken using a ZEISS Laser Scanning Microscope LSM 980 with Airyscan 2. e Electron microscopy to identify CLIC structures in GII.4 VLP-treated HIEs. (1–7 structures/cell) compared to, mock-treated cells (no structures in n = 25 cells). f Confocal microscopy to detect GII.4 VP1 capsid (green) colocalization with gal-3 (red) on the cell surface at 10 min and 1 h (37 o C) after VLP treatment using anti-gal-3 and Gp Syd-pAb (n = 3 HIE replicates). g Effect of blocking GII.4 virus-galectin-3 (gal-3) surface interaction using anti-gal-3 antibody on GII.4 replication at 1 h (black) and 24 h (red). h Dot blot analysis investigating GII.4 VLP interaction with purified gal-3. i Probing CLIC carriers utilized in HIEs for endocytosis with Alexa Fluor™ 594 conjugated cholera toxin B (CTxB) (red) and GII.4 VLPs (green) with similar cargoes marked by white arrows ( n = 2 HIE replicates). Inset: Co-occurrence of CTxB and GII.4 VLPs in similar cargos, scale = 5 µm. All the experiments were repeated independently three times with similar results. In b , c , and g viral GEs were quantified using n = 2 independent HIE replicates for the 1 h and n = 3 independent HIE replicates for 24 h with two technical replicates/sample. The error bars represent mean ± SD with P values calculated using one-way ANOVA, Dunnett’s multiple comparisons test with comparisons at 24 h relative to untreated control. Source data are provided as a Source Data file.

Article Snippet: ML141 , SCBT , sc-362768.

Techniques: Laser-Scanning Microscopy, Electron Microscopy, Confocal Microscopy, Blocking Assay, Virus, Dot Blot, Purification, Control

Journal: Nature Communications

Article Title: CLIC and membrane wound repair pathways enable pandemic norovirus entry and infection

doi: 10.1038/s41467-023-36398-z

Figure Lengend Snippet: List of inhibitors, additives and recombinant proteins

Article Snippet: ML141 , SCBT , sc-362768.

Techniques: Recombinant

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: Inhibition of Cdc42 in 3D biomimetic angiogenic model. (A) Schematic of our 3D biomimetic model of angiogenesis. A device is consisted of 2 channels fully embedded inside 2.5mg/ml collagen gel. (B) Cdc42 activity was reduced in half in the presence of 15 μM Cdc42 inhibitor ML141. (C) Representative phase images of sprouts guided by a gradient of angiogenic cocktail including MCP-1, VEGF, PMA, and S1P at Day 4 for control DMSO and Cdc42-inhibited devices. Average invading distance of invading cells into matrix was reduced in the presence of ML141 (N=4 individual experiments); * (p<0.05) indicates statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Inhibition, Activity Assay, Control

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: The effects of Cdc42 on sprout length and density during angiogenesis sprouting. (A) Quantification of sprout density between control DMSO and Cdc42 inhibition conditions. ML141 was initiated at onset of sprouting over a course of 4 days (n=4 individual experiments). The presence of Cdc42 inhibitor slightly decreased the sprout density. (B) Sprout length was quantified at day 4 using images acquired from confocal microcopy. Sprout length was halved when Cdc42 activity was partially inhibited (n=4 individual experiments). (C) Quantification of average sprout angle between DMSO and ML141 devices (n=4 individual experiments) revealed unaltered directional migration of the multicellular sprout structures. (D) Quantification of the number of invading cells demonstrated inhibition of Cdc42 reduced migrating cells into the interstitial matrix (n=4 individual experiments). (E) Representative 3D projections of Z-stack confocal images of sprouts in DMSO and ML141 conditions at day 4. White arrowheads indicate single migrating cells. Scale bar is 100 μm. (F) Quantification of single cell migration among migrating cells in the interstitial matrix revealed a significant increase in the fraction of single migrating cells (n=4 individual experiments). Unit area is 300 μm2. * indicates statistical significance (P<0.05); ns indicates no statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Control, Inhibition, Activity Assay, Migration

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: The effects of antagonizing Cdc42 on branching morphogenesis of angiogenic sprouting. (A) A schematic of two different branching structures (branch and intersegmental branch) observed in angiogenic sprouting in our model guided by a gradient of angiogenic cocktail. (B) Number of branch points is quantified for DMSO vs ML141 conditions. (C) The fraction of sprouts with branches was reduced in the presence of ML141. (D) The fraction of sprouts with intersegmental branches was also reduced when Cdc42 activity was perturbed with ML141. (E) Average length of branch was unaffected by the inhibition of Cdc42. (F) Average length of intersegmental branches was also unaffected by the inhibition of Cdc42. N=4 individual experiments; * (p<0.05) indicates statistical significance; ns indicates no statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Activity Assay, Inhibition

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: Cdc42 regulates branching in angiogenic sprouting in vitro

doi: 10.1111/micc.12372

Figure Lengend Snippet: Filopodia formation of endothelial cell sprouting upon Cdc42 inhibition. (A) Representative confocal images of phalloidin-stained sprout tip cells showing filopodia-like extensions in DMSO and ML141 conditions. Sprouting was initiated for 3 days. Then 22.5μM ML141 was added for 4hrs before fixation. (B) Average angle of filopodia of sprout tip cells remained unchanged upon inhibition of Cdc42 (n=4 individual experiments). (C) The number of filopodial extensions per sprout tip cells in DMSO and ML141 conditions (n=4 individual experiments) displayed a surge in filopodia-like extension in ML141 treatment. (D) Histogram showing distribution of the filopodia-like extension numbers per sprout tip cells for DMSO and ML141 conditions (n=4 individual experiments). (E) Average length of filopodia-like extensions is quantified for DMSO and ML141 conditions (n=4 individual experiments). (F) Histogram showing distribution of the length of the filopodia-like extensions for DMSO and ML141 conditions. * (p<0.05) and *** (p< 0.001) indicate statistical significance.

Article Snippet: Methods Using a 3D biomimetic model of angiogenesis in vitro , where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting.

Techniques: Inhibition, Staining

Journal: NPJ Parkinson's Disease

Article Title: Microglial low-affinity FcγR mediates the phagocytic elimination of dopaminergic neurons in Parkinson’s disease degeneration

doi: 10.1038/s41531-025-01249-9

Figure Lengend Snippet: A CD16/32 accumulates at the leading front of motile microglia. Top panel shows maximum intensity projection of a BV2 cell marked with CD16/32 and DAPI. Leading lamellas (left pole) and the uropod (opposite pole) indicate its direction. Bottom panel shows a 0.5 μm optical plane represented in a scale of 16 colors. Scale on the bottom (white represents the highest value and black the lowest). Leading front lamellas (1) and uropod (2) are indicated (Scale bar: 10 μm). B Illustration of a characteristic motile microglial cell showing essential cytoskeletal elements (Modified from Roig-Martinez et al.) . C Plot of relative fluorescence of motile microglia measured (white broken line) from the image on the left. Note the higher fluorescence at the leading front (1) and a smaller peak at the uropod (2). D BV2 microglia in kinetic shape showing accumulation of Cdc42 at the leading edge (asterisk). E Relative fluorescence of BV2 microglia from panel D indicates the Cdc42 increase at the cell frontal lamella (asterisk) (Scale bar: 10 μm). F Representative image of a BV2 microglial cell expressing a high density of Cdc42 (red) in an F-actin-rich protrusion. The nucleus was counterstained with DAPI (blue). (Scale bar: 3 μm). G Plot profile of relative fluorescence displays high expression of Cdc42 corresponding with high fluorescence of F-actin at the protruding edge (2) compared with the nucleus (1). H Clusters of Cdc42 can be seen at the microglial lamelipodia. Detailed of clusters are shown in higher magnification (yellow arrowheads) from white inset (Scale bar: 10 μm). I Cdc42 is spread as filaments towards to F-actin-rich microglial cell border. Higer magnification from white inset shows detail of the filaments (white arrowheads) (Scale bar: 10 μm). J Higher resolution images from orange inset show that Cdc42 accumulates at the inner part of the F-actin cellular processes (Z confocal steps +3 μm and +4 μm from the culture plate bottom are shown) (Scale bar: 5 μm). K Confocal image of microglial cell establishing a phagocytic cup around a dopaminergic cell (1). The PC12 DA cell is marked with dopaminergic neuronal marker, TH (green), and the microglial cell is stained by F-actin (magenta). (2) Diagram of the engulfing process depicted in 1. (3) Image of F-actin fluorescence intensity of the engulfing microglia seen in 1. Note the higher intensity is displayed at the borders of the phagocytic cup. (4) higher magnification of the phagocytic cup to visualize the characteristic accumulations of F-actin (Scale bar: 10 μm). (5) 3D reconstruction of the phagocytic event displaying the microglia arranging the F-actin-rich phagocytic cup around a dopaminergic cell (yellow arrowhead). L Increasing concentrations of ML141 were safe for BV2 cell viability. MTT cell viability assay for BV2 microglial cells submitted to increasing concentrations of ML141 was performed to test their safety: 500 μg of H 2 O 2 resulted in approximately 50% decrease of the cell viability. But no significant decrease was appreciated after the administration of ML141 or vehicle (*** p ˂0.001 H 2 O 2 significant against every other treatment). M Quantification of the number of PC12 DA cells at 60 min. of interaction with BV2 microglia. BV2 were treated either before or after activation with Cdc42 inhibitor ML141. The proinflammatory-mediated elimination of DA cells was prevented when BV2 cells were incubated with ML141. (Pre-activation inhibition ** p ˂0.01 IFN-γ + LPS vs. ML141/IFN-γ + LPS and ML141. Post-activation inhibition $$$ p ˂0.001 IFN-γ + LPS vs. every condition and **** p ˂0.0001 control vs. IFN-γ + LPS).

Article Snippet: To analyze how the inhibition of the protein Cdc42 affects the preservation of dopaminergic cells, the inhibitor ML141 (Cdc42/Rac1 GTPase Inhibitor, MERCK, Calbiochem, San Diego, CA, USA) was used in BV2/PC12 co-cultures.

Techniques: Modification, Fluorescence, Expressing, Marker, Staining, Viability Assay, Activation Assay, Incubation, Inhibition, Control

Journal: NPJ Parkinson's Disease

Article Title: Microglial low-affinity FcγR mediates the phagocytic elimination of dopaminergic neurons in Parkinson’s disease degeneration

doi: 10.1038/s41531-025-01249-9

Figure Lengend Snippet: A Diagram of the procedure used for the CD16/32 passive immunotherapy. Neuropathological histology was performed in the SNpc (red square). B Representative confocal images showing the SNpc labeled with TH and counterstained with DAPI. Depletion of DA neurons can be appreciated in MPTP-treated animals. However, a protective effect can be seen in mice treated with CD16/32 neutralizing monoclonal antibodies (αCD16/32) (Scale bar: 30 μm). C Quantification of the DA neurons in the SNpc demonstrating a significant decrease in the MPTP group which is prevented by the administration of CD16/32 (*** p ˂0.001 MPTP vs. every treatment except MPTP + Isotype, ### p˂0.001 MPTP + Isotype vs. every treatment except MPTP). D 3D reconstruction of representative microglial cells expressing Iba-1 in different treatments of the experiment (Scale bar: 10 µm). E Quantification of the area of Iba-1 in the SNpc, indicating variation of microglial size when the animals were intoxicated with MPTP. F CD16/32 immunostaining reveals increased immunoreactivity in MPTP-treated animals (Scale bar: 20 μm). G Quantification of CD16/32 cell number shows increased levels in MPTP-treated animals. H Diagram of the procedure used for Cdc42 inhibition. I Representative confocal images of histological analysis. Upper panel: Representative images of TH positive neurons of the SNpc in every treatment (Scale bar: 30 µm). Lower panel: 3D reconstructions illustrating morphology and changes of size in microglial cells expressing Iba-1 (Scale bar: 10 µm). J Quantification of TH positive neurons indicating a significant decrease in the MPTP group and prevention by the previous administration of Cdc42 inhibitor ML141 (** p < 0.01 with respect to saline). K Quantification of the Iba-1 expressing area, which represents changes in microglial size. Microglial activation is present in the MPTP group evidenced by the increased area of Iba-1, which is decreased in the animals intoxicated with MPTP but previously treated with ML141 (*** p < 0.001 with respect to controls, $$ p < 0.01 with respect to ML141). L CD16/32 immunostaining reveals immunoreactivity in the MPTP group, providing a strong inhibition in MPTP-ML141 group (Scale bar: 30 μm). M Quantification of CD16/32+ microglia show a significant increase in the MPTP-treated group (** p < 0.01 compared to ML141 + MPTP).

Article Snippet: To analyze how the inhibition of the protein Cdc42 affects the preservation of dopaminergic cells, the inhibitor ML141 (Cdc42/Rac1 GTPase Inhibitor, MERCK, Calbiochem, San Diego, CA, USA) was used in BV2/PC12 co-cultures.

Techniques: Labeling, Bioprocessing, Expressing, Immunostaining, Inhibition, Saline, Activation Assay