Fig 1: Identification of a target engagement antibody for RIPK1. (A) Comparison of RIPK1 antibodies (Ab1‐Ab7) with changes observed in HDX‐MS experiment between GSK’481‐bound protein and GSK’064‐bound protein. Ab3 is abcam 125072. Ab5 is Cell Signaling 3493. Ab3, Ab4, Ab6, and Ab7 are shown, though they do not overlap with the RIPK1 (1‐324) truncated protein used in HDX‐MS. These antibodies recognize a region at the RIPK1 C‐terminus from AA325‐671. The exact epitopes of these antibodies remain unknown. (B) HT‐29 cell lysates (20 μg) were incubated with GSK’481, GSK’253, or GSK’064 at concentrations ranging from 1 μmol·L−1 to 1 pmol·L−1 for 24 hours. Cell lysates were analyzed by immunoassay using Abcam ab72139 mouse monoclonal antibody for RIPK1 capture and Ab5 (Cell Signaling 3493) at 1 μg·mL−1 final concentration. Raw ECL counts were plotted against log molar concentrations of RIPK1 inhibitor. Samples were screened in single well format. (C) HT‐29 cell lysates (20 μg) were incubated with GSK’481, GSK’253, or GSK’064 at concentrations ranging from 1 μmol·L−1 to 1 pmol·L−1 for 24 hours. Cell lysates were analyzed by immunoassay using Abcam ab72139 mouse monoclonal antibody for RIPK1 capture and Ab3 (Abcam ab125072) at 1 μg·mL−1 final concentration. Raw ECL counts were plotted against log molar concentrations of RIPK1 inhibitor. Samples were screened in single well format. (D) Schematic representation of target engagement model demonstrating “competition” of FREE‐RIPK1 antibody by RIPK1 inhibitors of the BOAZ chemical class. ECL, Electrochemiluminescent