Extended Data Fig. 5: Related to Fig. 4.
From: PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase
a, Isothermal titration calorimetry experiments of AURORA-A into TBD (left panel, no measurable binding was observed), the binary complexes of AURORA-A/JB170, CEREBLON(TBD)/JB170 and ternary complex of AURORA-A/CEREBLON(TBD)/JB170. Shown are the raw heat rates on the top panel with integrated, baseline-corrected heats per injection and the corresponding fits below. An overlay of all curves (merged) is shown on the bottom left. b, Immunoblots of immunoprecipitation experiments. AURORA-A was precipitated with an anti-HA tag antibody from MV4–11 cells stably expressing HA-tagged AURORA-A or control cells transduced with an empty vector after treatment with 0.5 µM JB170 or 5 µM thalidomide (Thal.). The amount of co-precipitated CEREBLON and VHL was tested by immunoblotting. c, Immunoblots of immunoprecipitation experiments. AURORA-A was precipitated with an anti-HA tag antibody from MV4–11 cells stably expressing HA-tagged AURORA-A ( + ) or from control cells (-) after treatment with CEREBLON-based degraders (JB170, JB171, JB211), VHL-based degraders (JB160, JB161) or DMSO. The amount of co-precipitated CEREBLON and VHL was tested by immunoblotting. d, Immunoblots of immunoprecipitation experiments. BRD4 was precipitated with a specific antibody from MV4–11 cells after treatment with the VHL-based PROTAC MZ1 or DMSO in the presence of 5 mM MG132 for 1 h. The amount of co-precipitated VHL was tested by immunoblotting. e, Structural superposition of AURORA-A (blue, with bound alisertib in green) and AURORA-B. Residues with a maximum distance of 5 Å from CEREBLON in the AURORA-A-CEREBLON complex models are shown in cyan. Non-conserved amino acids near the proposed interaction interfaces of AURORA-A with CEREBLON are highlighted as labeled sticks and provided as a tabular list below the figure.
