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. 2015 Mar 19;57(6):1133–1141. doi: 10.1016/j.molcel.2015.02.012

Figure 4.

Figure 4

TopBP1 Does Not Protect BLM from Degradation

(A) TopBP1 depletion does not affect BLM levels. HeLa cells were transfected with the indicated siRNAs and harvested for western blotting 3 days later.

(B) Adenovirus-induced proteasomal degradation of TopBP1 does not affect BLM levels. U2OS cells were infected with the hr703 adenovirus and harvested at the indicated times for western blotting. E1A is a control for adenovirus infection.

(C) BLM-TopBP1 interaction does not maintain BLM stability. Cycloheximide (CHX) was added to U2OS cells stably expressing GFP-BLM proteins for the indicated times before harvesting for western blotting. The graph shows the level of BLM at the time points indicated as a percentage of untreated (0 h). Quantification was performed using ImageJ.

(D) Sequence alignment showing the evolutionary conservation of the BLM region containing Lys38, Lys39, and Lys40.

(E) Mutation of BLM Lys38, Lys39, and Lys40 to alanine (K3A) disrupts binding to TOP3A and RMI2. 293FT cells were transfected with the indicated plasmids and harvested for pull-downs 24 hr later. See also Figure S4.