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. Author manuscript; available in PMC: 2015 Apr 10.
Published in final edited form as: Mol Cell. 2014 Mar 20;54(1):107–118. doi: 10.1016/j.molcel.2014.02.017

Figure 3. The UBZ4 domain of ZBTB1 is required for targeting to UV damage sites and for PCNA monoubiquitination.

Figure 3

(A) Purified GST or GST-Ub was incubated with 293T lysates expressing GFP-tagged ZBTB1 wild-type or UBZ4 mutant (C536A & C539A), and analyzed by immunoblot and Ponceau S staining. (B) Anti-PCNA immunostaining in U2OS cells, transfected with GFP-tagged Rev1, Pol η, or ZBTB1 and treated with 100 J/m2 UVC for 2 hr through a 5 μm micropore filter. GFP-tagged Rev1 and Pol η were served as positive controls. Scale bar: 5 μm. (C) Anti-CPD immunostaining of U2OS cells transfected with GFP-ZBTB1 wild-type or mutant treated with UVC through micropore filter. ΔBTB: aa1-142 deletion. (D) Quantification of cells in (C), showing the percentage of cells with GFP-ZBTB1 colocalizing with CPDs among cells displaying positive anti-CPD signal. (E) Immunoblot analysis of siRNA-treated HeLa cells stably expressing siRNA-resistant ZBTB1 variants. Note that cells displaying high expression of the unstable UBZ4 mutant were chosen for achieving similar expression among variants. See also Figure S3.