Fig. 4

Differential p38δ:PROTAC:VHL complex affinities result in distinct cellular ubiquitination. a Both SJFα and SJFδ pull-down p38δ in vitro, however, SJFδ maintains ternary complex at lower concentrations. As in Fig. 3a, GST-tagged VBC was used as a “bait” to trap recombinant p38δ in the presence of vehicle (DMSO) or the indicated concentrations of SJFα or SJFδ. The rightmost lane represents a 1:25 dilution of initial input protein used in each pull-down. b SJFδ forms a more stable ternary complex with endogenous p38δ and VHL than does SJFα. GST-VBC was immobilized on glutathione sepharose beads and incubated with MDA-MB-231 whole-cell lysate (WCL) in the presence of vehicle (DMSO) or the indicated concentrations of SJFα or SJFδ. Beads were washed and “trapped” proteins (i.e., those that engage in a ternary complex) were eluted with SDS sample buffer and separated by SDS-PAGE. Samples were assessed by western blot and CUL2 and α-tubulin serve as positive and negative GST-VBC immunoprecipitation controls, respectively. c Only SJFδ ubiquitinates FLAG-p38δ in HeLa cells. As in Fig. 3c, HeLa cells were co-transfected with HA-Ub and FLAG-p38δ and were either treated with vehicle (DMSO), 1 µM SJFα, or 1 µM SJFδ for 2 h. FLAG-immunoprecipitated lysates were separated by SDS-PAGE and assessed via western blots detecting HA (Ub). Smears represent ubiquitin-conjugated FLAG-p38δ and numerical markers to the left of the western blot refer to kilodalton (kDa) masses. See Supplementary Figures 5 and 6 for additional p38δ:PROTAC:VHL characterization