Extended Data Fig. 6: Related to Fig. 5.
From: PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase
a, Histogram of BrdU incorporation. The amount of incorporated BrdU is shown for cells in the S-phase in Fig. 5a. b, Histogram of BrdU incorporation. The amount of incorporated BrdU is shown for cells in the S-phase in Fig. 5e. c, Immunoblot of AURORA-A. IMR5 cells overexpressing HA-tagged AURORA-A upon incubation with doxycycline (Dox) or control cells (EtOH) were treated with JB170 (1 µM) or alisertib (1 µM) for 18 h. d, Flow cytometry plots showing cell cycle distribution. IMR5 cells overexpressing AURORA-A upon incubation with doxycycline (Dox) or control cells (EtOH, as shown in Extended Data Fig. 6c) were treated with JB170 (1 µM) or alisertib (1 µM) for 18 h. Cells were labeled with BrdU, stained with PI, and analyzed by flow cytometry. The amount of intercalating PI (top) and the correlation of BrdU to PI (bottom) are shown. e, Histogram showing BrdU incorporation for cells in the S-phase in Extended Data Fig. 6d. f, Immunoblot of AURORA-A. AURORA-A was depleted in IMR5 cells by an siRNA and AURORA-A levels were analyzed by immunoblotting for biological triplicates. Cells were analyzed by flow cytometry as shown in Extended Data Fig. 6g,h. g, Cell cycle distribution analyzed by flow cytometry. IMR5 cells depleted for AURORA-A by siRNA were labeled with BrdU, stained with PI, and analyzed by flow cytometry. The amount of intercalating PI (top) and the correlation of BrdU to PI (bottom) are shown. h, Histogram showing BrdU incorporation for cells in the S-phase in Extended Data Fig. 6g and two additional biological replicates. i, Scatter plot of the AURORA-A interactome. The X-axis displays the enrichment (log2FC) of proteins in HA-AURORA-A-expressing cells compared to control cells (Ctr). The Y-axis displays the protein intensities (log10). Substrates of AURORA-A5 are labeled. j, Immunoblots of HA-tagged AURORA-A and DICER1. HA-precipitations were performed from HEK293 cells transfected to express versions of HA-tagged catalytically inactive AURORA-A (AURORA-AD274N, AURORA-AK162R) or the wild-type protein. Exogenous AURORA-A was detected with an anti-HA tag antibody, and levels of DICER1 were analyzed in the input and immunoprecipitant by antibodies recognizing the endogenous protein. Control cells (Ctr) did not express HA-tagged protein. k, AURORA-A sedimentation profile in the presence and absence of RNases. The graph is a re-analysis of published data46 retrieved from http://r-deep.dkfz.de/.
