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. Author manuscript; available in PMC: 2013 Apr 2.
Published in final edited form as: Mol Cell. 2012 Aug 10;47(3):396–409. doi: 10.1016/j.molcel.2012.05.024

Figure 2. Interaction of ZRANB3 with ubiquitinated PCNA in vitro.

Figure 2

(A) Sequence alignment of the NZF motifs of ZRANB3, TAB2, TAB3, ZRANB1 and RANBP2. Residues involved in the binding of proximal and distal ubiquitin binding sites, as described in (Kulathu et al., 2009), are within green and blue boxes, respectively. NZF motif residues that have been mutated are indicated.

(B) Association of K63-linked ubiquitin chains with the ZRANB3 NZF motif. GST-ZRANB3 containing a WT or mutant NZF motif were affinity purified from bacteria and incubated with mono-ubiquitin or ubiquitin chains linked on lysine 48 (K48) or lysine 63 (K63). Complexes were detected with anti-ubiquitin and anti-GST antibodies after western blotting.

(C) Association of ZRANB3 with ubiquitinated PCNA. WT or mutant FLAG-ZRANB3 was immunoprecipitated with anti-FLAG beads from insect cells and incubated with a mixture of unmodified, mono- and poly-ubiquitinated PCNA. Immunoprecipitated PCNA and FLAG-ZRANB3 were detected by western blotting.