Fig. 4

OligoTRAFTACs induce brachyury-GFP degradation via the proteasomal pathway. (A) HEK293T cells expressing brachyury-GFP were transfected with 75 nM each of OT3 and its scrambled OT6, and lysates were probed for brachyury levels. (n = 2, **p < 0.005) (B) OT3 induced brachyury degradation is VHL-dependent. HEK293T cells were preincubated with and without 10 μM of VHL ligand for 1.5 h prior to OT3 transfection. After 20 h of transfection, cells lysates were prepared and analyzed for brachyury degradation. (n = 3, ****p < 0.0001) (C) OT3 induces brachyury degradation via the proteasomal pathway: neddylation inhibitor MLN-4924 was preincubated with cells prior to OT3 transfection. After 20 h of transfection of OT3, cells were harvested and analyzed for brachyury levels. (n = 2, **p < 0.01) (D) Brachyury-GFP downregulation was monitored by GFP fluorescence in cells in the presence or absence of MLN-4924. (n = 2).