Fig. 4. In-gel fluorescence, immunoblot and proteomics analysis by competitive ABPP in HEK293 cells (a) general workflow for competitive ABPP. (b) In-gel fluorescence analysis of competitive ABPP of MT16-001 and MT16-009 against ABP MT16-205 in HEK293 cells. Protein loading was assessed by Coomassie blue staining (c) the ABP-labeled proteins were enriched using streptavidin magnetic beads. The total lysate before pulldown (TL) and pulldown (PD) samples were analyzed by immunoblotting using the indicated antibodies. HSP90 was used as a protein loading control. (d) The labeled proteins were analyzed by nanoLC-MS/MS. The data was processed in MaxQuant and analyzed in Perseus. Data represent mean ± SEM (three biological replicates). The hits were selected in the ABP-enriched proteins with the fold-change >4 and p-values >1. The selected hits were normalized to vehicle controls.