Fig. 3. DB008 covalently modifies Cys169 of PARP16 and exhibits excellent proteome-wide selectivity in the irreversible binding mode. (A) Model of DB008 covalently bound to C169 of PARP16. (B) HEK 293T cells were transfected with Myc2x-tagged PARP16 WT or the C169S mutant, treated with a DB008 dose response for 2 h, followed by lysis and clicking to TAMRA-azide for in-gel fluorescence detection of PARP16 labeling. (C) Quantification of TAMRA signal from (B); n = 3 biological replicates. (D) HEK 293T cells were transfected with Myc2x-tagged PARP16 WT, treated with a 300 nM DB008 on a time course, followed by lysis and clicking to TAMRA-azide for in-gel fluorescence detection of PARP16 labeling. (E) Quantification of TAMRA signal from (D); n = 3 biological replicates. (F) DB008 proteome-wide labeling in HAP1 cells. HAP1 WT and HAP1 PARP16 KO cells were treated with a DB008 dose response for 2 h, followed by lysis and clicking to TAMRA-azide for in-gel fluorescence detection of endogenous PARP16 labeling. (G) Quantification of TAMRA signal from (F); n = 2 biological replicates.