Extended Data Fig. 7: Validation of DCAF16 mutants.
From: Template-assisted covalent modification underlies activity of covalent molecular glues
a. Flow cytometry analysis of BRD4BD2-eGFP degradation in DCAF16 knockout K562 cells transduced with indicated HA-DCAF16 mutants and treated with increasing concentrations of KB02-JQ1 for 16 h (n = 3). b. Flow cytometry analysis of eGFP-SPIN4 stability in DCAF16 knockout K562 cells transduced with indicated HA-DCAF16 mutants (n = 3). c. Western blots of Flag immunoprecipitation (IP) from 293 T cells transfected with indicated HA-DCAF16 mutants and Flag-SPIN4 constructs. d. Close up of DCAF16 Ala53 orienting towards the hydrophobic core. e. Close up of DCAF16 Cys177 and Cys179 coordinating a structural zinc ion. f. Intact protein mass spectra of DDB1-DCAF16(WT) co-incubated with MMH2, DDB1-DCAF16(WT) co-incubated with MMH2 and BRD4BD2, DDB1-DCAF16(C58S) co-incubated with MMH2, or DDB1-DCAF16(C58S) co-incubated with MMH2 and BRD4BD2 at 4 °C for 16 h. All western blot data are representative of two independent measurements.
