Figure 6. Screen for CRL-mediated ubiquitination events in the DDR identifies EXO1 as an SCF substrate.
(A) Diagram of approach to identify UV or IR induced ubiquitination events mediated by CRLs. Light cells were treated with 10 ιM MLN4924 and 5 ιM MG132 for 30 minutes while heavy cells were treated with 5 ιM MG132 for 30 minutes. Both heavy and light plates were then irradiated with 40 J/m2 UV or 10 Gy IR and harvested 1 hour later.
(B) Correlation coefficients for Log2(L/H) ratios for 3 biological replicates identifying CRL substrates in the presence of UV and for 2 biological replicates in the presence of IR.
(C) Overlap of sites and proteins ubiquitinated more than 2-fold in response to UV (from UV, UV-MG, and UV-SCX datasets) with sites whose ubiquitination is suppressed more than 2-fold by MLN4924 (from UV-MLN dataset).
(D) Identification of proteins whose ubiquitination is stimulated by UV and inhibited by MLN4924. Blue bars represent Log2(L/H) ratios of diGly sites from UV-MG132 dataset (Figure 2A) while red bars represent ratios from UV-MLN dataset (Figure 6A). EXO1 represents a previously uncharacterized CRL substrate.
(E) (Above) HeLa cells were released from a double-thymidine block and harvested at 2 hour intervals. Two hours prior to each point of harvest, cells were treated with 1 ιg/mL 4NQO. Propidium iodide stained cells from each time point were analyzed by flow cytometry, and lysates blotted for EXO1. (Below) HeLa cells were released from a double thymidine arrest and harvested 6 hours later. At 4 hours prior to harvest, they were treated with 5 ιM MLN4924 for 2 hours followed by 1 ιg/mL 4NQO for 2 hours.
(F) HeLa cells infected with lentiviruses expressing dominant negative Cullins or empty vector (EV) were harvested 2 hours after 1 ιg/mL 4NQO treatment and blotted for EXO1. See also Figure S5.