rhoa pulldown activation assay kit  (Cytoskeleton Inc)

Journal: bioRxiv

Article Title: Microglial lipid signaling drives glioblastoma invasion and represents a therapeutic vulnerability

doi: 10.64898/2026.04.24.720633

Figure Lengend Snippet: (A) Gene expression of Yap-target genes ( Ankrd1, Ctgf, Cyr61 ) in NSCG (left) and NFpp10 (right) cells upon 24 hours of treatment with BV2 CM, BV2 CM LRA (lipid removal), BV2 CM LRA (lipid removal) LPA 18:1. Relative gene expression to S18. Fold increase. NSCG: n=3, 2way ANOVA, ** p-value<0.0056; *** p-value<0.0003, **** p-value<0.0001; NFpp10: n=3, 2way ANOVA, * p-value<0.0381, ** p-value=0.0012, *** p-value=0.0002, **** p-value<0.0001. (B) Representative immunoblots of Yap nuclear and cytoplasmic levels in NFpp10 cells upon 5 minutes, 10 minutes, 20 minutes of co-culture with BV2 cells and LPA 16:0 treatment. (C) Representative immunoblots of RhoA pulldown and total in NSCG (left) and NFpp10 (right) cells upon 2 minutes, 5 minutes, 10 minutes co-culture with BV2 cells. (D) Representative immunoblots of RhoA pulldown and total in NSCG (left) and NFpp10 (right) cells upon 5 minutes, 10 minutes co-culture with BV2 cells and LPA 18:1 or LPA 16:0 treatment respectively. (E) Invasion assays of NSCG (left) and NFpp10 (right) Scr (control) and YAP/TAZ knock-out cells in the presence or absence of BV2 cells. Fold increase. NSCG: n=3, One-way ANOVA, * p-value=0.0267, *** p-value=0.0001; NFpp10: n=6, One-way ANOVA, * p-value=0.0432, ** p-value=0.0034. (F) Invasion assays of NSCG Scr (control) and YAP knock-out cells in the presence or absence of BV2 cells and LPA 18:1 treatment. Fold increase. NSCG: n=3, One-way ANOVA, ** p-value<0.0022, *** p-value=0.0004, **** p-value<0.0001. (G) Tumor cell number across the 50 distinct TMAs. h. Ratio tumor cell/microglia-macrophages across the selected 15 tumor core and 15 tumor rim TMAs based on tumor cell density. i. Correlation tumor cells/microglia-macrophages across the selected 15 tumor core and 15 tumor rim TMAs. Each dot/number refers to a TMA sample. Correlation index: −0.477. All data represent mean ± SEM.

Article Snippet: Protein extraction was performed using the RhoA Pulldown activation Assay Kit (Cytoskeleton, BK036).

Techniques: Gene Expression, Western Blot, Co-Culture Assay, Control, Knock-Out

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , RhoA pull-down activation assay Biochem kit , Cytoskeleton , BK036-S , N/A.

Techniques: Purification, In Vitro, In Vivo, Recombinant, Enzyme-linked Immunosorbent Assay, Activation Assay, Plasmid Preparation, Sequencing

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) GTP-RhoA pull-down assay to detect the active level of RhoA in sgControl, sg MyD88, and sg TRAF6 HEK293T cells after 1 ng/mL IL-1β treatment for 30 min. Immunoblots showing activated RhoA and total RhoA. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of active RhoA versus total RhoA. ( B ) Representative confocal images of GFP-AHPH after 1 ng/mL IL-1β treatment for 30 min in sgControl, sg MyD88, and sg TRAF6 HEK293T cells. Scale bars, 10 μm. ( C ) Quantification of fluorescence intensity of GFP-AHPH in (B). Data are representative of eight individual repeats. ( D ) Representative confocal images of GFP-AHPH localization with or without 1 ng/mL IL-1β treatment at different time points of syncytia formation in HEK293T-S-HA and HEK293T-ACE2 cells. Schematics with green dots in the white dashed line boxes representing GFP-AHPH, red cycles representing S-expressing cells, and magenta cycles representing ACE2-expressing cells. White arrowheads indicate the localization of GFP-AHPH, scale bars, 10 μm. Images are representative of three independent experiments. ( E ) Representative confocal images of F-actin stained with phalloidin-488 in transfected vector or 20 ng RhoA-CA HEK293T-S-HA and HEK293T-ACE2 cells. Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells, and magenta cycles representing ACE2-expressing cells. Scale bars, 10 μm. Images are representative of three independent experiments. ( F ) Representative confocal images of F-actin stained with phalloidin-488 in the presence or absence of 1 ng/mL IL-1β or 50 μM Y-27632 treatment at different time points of syncytia formation in HEK293T-S-HA and HEK293T-ACE2 cells. Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells, and magenta cycles representing ACE2-expressing cells. White arrowheads (E and F) indicate the enrichment or disappearance of F-actin, scale bars, 10 μm. Images are representative of three independent experiments. Figure 4—source data 1. Annotated, uncropped blots in . Figure 4—source data 2. Raw, uncropped blots in . Figure 4—source data 3. Source data of the individual points in .

Article Snippet: Commercial assay or kit , RhoA pull-down activation assay Biochem kit , Cytoskeleton , BK036-S , N/A.

Techniques: Pull Down Assay, Western Blot, Fluorescence, Expressing, Staining, Transfection, Plasmid Preparation

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Single-channel confocal images of . ( B ) Luciferase activity (relative luminescence units [RLU]) measured from HEK293T cell lysates and immunoblots showing full-length spike, S2, cleaved S2’, ACE2, and Myc-RhoA collected from transfected vector, 10 or 20 ng constitutively active RhoA mutant (RhoA-CA) both in HEK293T-S and in HEK293T-ACE2 cells. Data and blots are representative of four individual repeats. Numbers below the blots indicated the intensity of S2’ versus Tubulin. ( C ) Single-channel confocal images of (top panel). ( D ) Single-channel confocal images of (middle panel). Figure 4—figure supplement 2—source data 1. Annotated, uncropped blots in . Figure 4—figure supplement 2—source data 2. Raw, uncropped blots in . Figure 4—figure supplement 2—source data 3. Source data of the individual points in .

Article Snippet: Commercial assay or kit , RhoA pull-down activation assay Biochem kit , Cytoskeleton , BK036-S , N/A.

Techniques: Luciferase, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Mutagenesis

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Representative confocal images of GFP-AHPH localization with or without 1 ng/mL interleukin-1β (IL-1β) treatment in 0.5 multiplicity of infection (MOI) wild-type (WT) authentic SARS-CoV-2-infected HEK293T-ACE2 cells at 6 and 24 hr post-infection (hpi). Schematics with green dots in the white dashed line boxes representing GFP-AHPH, red cycles representing SARS-CoV-2-infected cells, and white cycles representing neighboring cells. White arrowheads indicate the localization of GFP-AHPH, scale bars, 10 μm. Images are representative of three independent experiments. ( B ) Representative confocal images of F-actin stained with phalloidin-488 in the presence or absence of 1 ng/mL IL-1β treatment upon 0.5 MOI WT authentic SARS-CoV-2 infection of Caco-2 cells at 24 hpi. Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing SARS-CoV-2-infected cells, and white cycles representing neighboring cells. Scale bars, 10 μm. Images are representative of three independent experiments. ( C ) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’, N, and Myc-RhoA collected from HEK293T-ACE2 cells, which were transfected with vector, 10 or 20 ng RhoA-CA before infection with 0.5 MOI authentic SARS-CoV-2 WT strain for 24 hr. Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. ( D ) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’, N, and Myc-RhoA collected from lentivirus-transduced Calu-3 cells expressing vector or RhoA-CA, infected with WT authentic SARS-CoV-2 for 24 hr. Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. ( E ) Representative confocal images of F-actin stained with phalloidin-488 from Calu-3 cells described in (D). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells, scale bars, 10 μm. Images are representative of four independent experiments. ( F, G ) Immunoblots of WT SARS-CoV-2 S, S2, cleaved S2’, and N collected from Calu-3 cells (F) or primary human lung cells (G), which were infected with authentic SARS-CoV-2 for 1 hr, then washed with phosphate buffered saline (PBS) before being treated with different concentrations of Y-27632 and 10 ng/mL IL-1β for 24 hr. Blots are representative of three independent experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. ( H ) Representative confocal images of F-actin stained with phalloidin-488 in Calu-3 cells described in (F). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells. White arrowheads (B, E, and H) indicate the enrichment or disappearance of F-actin, scale bars, 10 μm. Images are representative of four independent experiments. Figure 5—source data 1. Annotated, uncropped blots in . Figure 5—source data 2. Raw, uncropped blots in .

Article Snippet: Commercial assay or kit , RhoA pull-down activation assay Biochem kit , Cytoskeleton , BK036-S , N/A.

Techniques: Infection, Staining, Western Blot, Transfection, Plasmid Preparation, Expressing, Saline

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: ( A ) Immunoblots of wild-type (WT) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) S, S2, cleaved S2’, N, and Myc-RhoA collected from Caco-2 cells, which were transfected with vector, 10 or 20 ng RhoA-CA before infection with 0.5 multiplicity of infection (MOI) WT authentic SARS-CoV-2 for 24 hr. Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S2’ or N versus β-Actin. ( B ) Representative confocal images of F-actin stained with phalloidin-488 from Caco-2 cells described in (A). Schematics with green lines in the white dashed line boxes representing actin bundles, red cycles representing S-expressing cells. White arrowheads indicate the enrichment or disappearance of F-actin, scale bars, 10 μm. Images are representative of four independent experiments. ( C ) Single-channel confocal images of (B). ( D ) Single-channel confocal images of . ( E ) Co-immunoprecipitation (IP) and input controls of full-length spike protein after anti-V5 or anti-IgG pull-down from cell lysates mixed between HEK293T cells expressing ACE2-V5-6his or Spike protein with vector or RhoA-CA (10 ng). Blots are representative of three individual experiments. Numbers below the blots indicated the intensity of S or S2 versus ACE2 in IP group and S, S2, or ACE2 versus Tubulin in input group. ( F ) Representative confocal images of wheat germ agglutinin (WGA, cell surface marker) and ACE2 from HEK293T cells co-transfected with ACE2 and vector or RhoA-CA (10 ng). Scale bars, 10 μm. Images are representative of three independent experiments. ( G ) Quantification of the relative ACE2 expression on the cell surface in (F). Figure 5—figure supplement 2—source data 1. Annotated, uncropped blots in . Figure 5—figure supplement 2—source data 2. Raw, uncropped blots in . Figure 5—figure supplement 2—source data 3. Source data of the individual points in .

Article Snippet: Commercial assay or kit , RhoA pull-down activation assay Biochem kit , Cytoskeleton , BK036-S , N/A.

Techniques: Western Blot, Transfection, Plasmid Preparation, Infection, Staining, Expressing, Immunoprecipitation, Marker

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet: Host factors secreted from innate immune cells upon Toll-like receptor (TLR) ligands stimulation, including interleukin-1β (IL-1β) and IL-1α, act on both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells expressing spike protein and neighboring cells expressing ACE2 receptor via IL-1R1-MyD88-IRAK-TRAF6 signaling pathway, which leads to strong enrichment of activated RhoA at cell-cell junction, resulting in the formation of ROCK-mediated actin bundle to prevent SARS-CoV-2-induced cell-cell fusion and further viral transmission through syncytia formation.

Article Snippet: Commercial assay or kit , RhoA pull-down activation assay Biochem kit , Cytoskeleton , BK036-S , N/A.

Techniques: Infection, Expressing, Transmission Assay

Journal: eLife

Article Title: Interleukin-1 prevents SARS-CoV-2-induced membrane fusion to restrict viral transmission via induction of actin bundles

doi: 10.7554/eLife.98593

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , RhoA pull-down activation assay Biochem kit , Cytoskeleton , BK036-S , N/A.

Techniques: Purification, In Vitro, In Vivo, Recombinant, Enzyme-linked Immunosorbent Assay, Activation Assay, Plasmid Preparation, Sequencing

Journal: Nature communications

Article Title: An allosteric inhibitor of RhoGAP class-IX myosins suppresses the metastatic features of cancer cells.

doi: 10.1038/s41467-024-54181-6

Figure Lengend Snippet: Fig. 4 | Adhibin causes Myo9b misdistribution and interferes with RhoA- signalling. A Representative z-projections showing actin and active RhoA (colour- coded dtomato-rothekin intensity, scale bars: 20 μm) and (B) Myo9b and actin localization in adhibin-treated B16-F1 cells (scale bars: 10 μm). C z-Projections showing localization of Myo9b in B16-F1 cells before and after adhibin treatment. The red line defines the distance from rear to front. Scale bars: 20 μm. D Intensity profile of Myo9b along the rear to front section of B16-F1 cells in the presence and absence of adhibin. E pMLC2 and actin localization in 1% DMSO and 5 μM adhibin treated B16-F1 cells. Scale bars: 10 μm. F z-Projections of native and Myo9b-eGFP-

Article Snippet: Additionally, the RhoA PullDown Activation Assay Biochem Kit (Bead Pull-Down Format, Cytoskeleton: BK036, shown in Fig. S4Q) was used according to the manufacturer’s instructions to measure active RhoA.

Techniques:

Journal: Nature communications

Article Title: An allosteric inhibitor of RhoGAP class-IX myosins suppresses the metastatic features of cancer cells.

doi: 10.1038/s41467-024-54181-6

Figure Lengend Snippet: Fig. 6 | Adhibin interferes with RhoA-mediated activation of NM2 contractility. A, B Representative sum-projections showing actin and pMLC2 localization in adhibin-treated A459 and MLE-12 cells. Scale bars: 20 μm. C, D Amount of pMLC2 in control and adhibin-treated A459 cells, and MLE-12 cells (two-tailed Mann–Whitney test for two-column comparison, or one-way Kruskal–Wallis test for multiple

Article Snippet: Additionally, the RhoA PullDown Activation Assay Biochem Kit (Bead Pull-Down Format, Cytoskeleton: BK036, shown in Fig. S4Q) was used according to the manufacturer’s instructions to measure active RhoA.

Techniques: Activation Assay, Control, Two Tailed Test, MANN-WHITNEY, Comparison