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Validation of CL16-RhoA binding by LC-MS/MS experiments (A) Workflow of LC-MS/MS experiments to detect RhoA-CL16 engagement in <t>HCT116</t> 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.
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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.

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: Human: HCT116 (Wildtype/48Y/Male) , ATCC , #CCL-247.

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