Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Identification of CL16 binding to RhoA by gel-based ABPP experiment (A) Rationale of gel-based ABPP for screening compound binding to RhoA in vitro . Purified RhoA protein was pre-treated with solvent control or library compounds followed by labelling with fluorescent activity-based probes. (B) Expected outcomes in gel-based ABPP. In the 1 st round of screening, compound binding to RhoA results in a decrease in in-gel fluorescence (FL) intensity. Further validation of the binding can be performed by experiments using varying concentrations of the hit compounds. In the 2 nd round of screening, selected hits are examined for their binding to Cdc42 and Rac1 to determine their selectivity for RhoA.

Article Snippet: Rac1 Pull-Down Activation Assay Biochem Kit , Cytoskeleton , Cat# BK035.

Techniques: Binding Assay, In Vitro, Purification, Solvent, Control, Activity Assay, Fluorescence, Biomarker Discovery

Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Biochemical assays to Investigate RhoA inhibition by CL16 (A) Expected results of RhoGTPase activity assay in which CL16 selectively impairs the activation of RhoA and not Rac1 and Cdc42. (B) Schematic diagram illustrating the workflow of the co-immunoprecipitation experiment. (C) Expected results of co-immunoprecipitation whereas CL16 interferes with interactions between RhoA and ARHGEF1.

Article Snippet: Rac1 Pull-Down Activation Assay Biochem Kit , Cytoskeleton , Cat# BK035.

Techniques: Inhibition, Activity Assay, Activation Assay, Immunoprecipitation

Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Identification of CL16 binding to RhoA by gel-based ABPP experiment (A) Rationale of gel-based ABPP for screening compound binding to RhoA in vitro . Purified RhoA protein was pre-treated with solvent control or library compounds followed by labelling with fluorescent activity-based probes. (B) Expected outcomes in gel-based ABPP. In the 1 st round of screening, compound binding to RhoA results in a decrease in in-gel fluorescence (FL) intensity. Further validation of the binding can be performed by experiments using varying concentrations of the hit compounds. In the 2 nd round of screening, selected hits are examined for their binding to Cdc42 and Rac1 to determine their selectivity for RhoA.

Article Snippet: RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) , Cytoskeleton , Cat# BK036.

Techniques: Binding Assay, In Vitro, Purification, Solvent, Control, Activity Assay, Fluorescence, Biomarker Discovery

Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Validation of CL16-RhoA binding by LC-MS/MS experiments (A) Workflow of LC-MS/MS experiments to detect RhoA-CL16 engagement in HCT116 cells. CL16-treated cells were harvested, digested and analyzed by LC-MS/MS. (B) Representative MS/MS showing CL16 modification on RhoA Cys16 in HCT116 cells. (C) Workflow of LC-MS/MS experiments using CL16-alkyne (a CL16-molecular probe) to study target profile of CL16. (D) Volcano plot revealing protein targets of CL16 identified by CL16-molecular probe. Statistical analyses were performed by two-tailed Student’s t-test by MS Excel. (E) Venn diagram summarizing the protein targets of CL16 identified in (A) and (C), highlighting RhoA as the primary target.

Article Snippet: RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) , Cytoskeleton , Cat# BK036.

Techniques: Biomarker Discovery, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Modification, Two Tailed Test

Journal: STAR Protocols

Article Title: Protocol to identify covalent inhibitors targeting RhoA Cys16

doi: 10.1016/j.xpro.2026.104494

Figure Lengend Snippet: Biochemical assays to Investigate RhoA inhibition by CL16 (A) Expected results of RhoGTPase activity assay in which CL16 selectively impairs the activation of RhoA and not Rac1 and Cdc42. (B) Schematic diagram illustrating the workflow of the co-immunoprecipitation experiment. (C) Expected results of co-immunoprecipitation whereas CL16 interferes with interactions between RhoA and ARHGEF1.

Article Snippet: RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) , Cytoskeleton , Cat# BK036.

Techniques: Inhibition, Activity Assay, Activation Assay, Immunoprecipitation