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. Author manuscript; available in PMC: 2016 Mar 9.
Published in final edited form as: Dev Cell. 2015 Feb 26;32(5):589–603. doi: 10.1016/j.devcel.2015.01.024

Figure 5. Ubiquitylation of CENP-A by the CUL4A-RBX1 Complex Enhances the Binding to HJURP In Vitro.

Figure 5

(A) In vitro GST pull-down assay was performed using purified proteins after CENP-A ubiquitylation in vitro. In vitro ubiquitylation was performed as in Figure 2E and subsequently purified GST-HJURP-6xHis or GST was added to perform in vitro GST pull-down assay. Proteins in 3% of the total reaction (Input) and pulled down with glutathione Sepharose (P) were detected (see Protein Purification and In Vitro GST Pull-Down Assay in Supplemental Experimental Procedures).

(B) In vitro immunoprecipitation was performed using purified proteins after CENP-A ubiquitylation in vitro. In vitro ubiquitylation was performed as Figure 2E and subsequently purified GST-HJURP-6xHis or GST was added to perform immunoprecipitation. Proteins in 3% of the total reaction (Input) and immunoprecipitates (IP) obtained were detected (see Immunoprecipitation Assay in Supplemental Experimental Procedures).