Fig. 3
From: Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase
p38α selectivity is discriminated by ternary complex formation. a Only p38α incubated in the presence of SJFα can be immunoprecipitated with GST-tagged VHL/EloB/EloC (VBC). Immobilized VBC was used as a “bait” to trap purified p38α in the presence of vehicle (DMSO), SJFα, or SJFδ (represented in micromolar concentrations). The rightmost lane represents a 1:25 dilution of initial input protein used in each pull-down. b Proximity-based AlphaLISA assay detects significant p38α:SJFα:VHL ternary complex, but no p38α:SJFδ:VHL ternary complex. His-p38α and GST-VBC were incubated in the presence of increasing concentrations of SJFα and SJFδ and the extent of ternary complex formation was assessed by excitation with incident light with λ = 680 nm and capture of the emission light at λ = 615 nm. Error bars represent the s.d from quadruplicate experiments. c Only SJFα ubiquitinates FLAG-p38α in HeLa cells. HeLa cells co-transfected with HA-Ubiquitin (HA-Ub) and FLAG-p38α were subsequently treated with either vehicle (DMSO), 500 nM SJFα, or 500 nM SJFδ for 1 h. FLAG-immunoprecipitated lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and assessed via western blots detecting HA (Ub). Smears represent ubiquitin-conjugated FLAG-p38α and numerical markers to the left of the western blot refer to kilodalton (kDa) masses. WCL refers to “whole-cell lysate” input
