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. Author manuscript; available in PMC: 2012 Jun 14.
Published in final edited form as: Biochem J. 2010 Jul 1;429(1):127–136. doi: 10.1042/BJ20091461

Figure 5. Ubiquitin can modify LDB1 at Lys-134.

Figure 5

(A) HEK293 cells were transfected with expression vectors for LDB1-FLAG (Ldb1), poly-His-Ubiquitin (His-Ub) and AU1-RNF12 (RNF12) as indicated. At 20 h after transfection, whole cell extracts were prepared and His-containing proteins were isolated under denaturing conditions by Ni++-chelate chromatography. An aliquot of the whole-cell extract (5% input) or the proteins isolated by Ni++-chelate chromatography was resolved by denaturing gel electrophoresis. FLAG-tagged LDB1 was visualized by immunoblotting with FLAG monoclonal antibody. Proteins with the appropriate migration for full-length, FLAG-tagged LDB1 indicated with a closed arrow and a more slowly migrating form of LDB1 is indicated with an open arrow. (B) HEK293 cells were transfected with expression vectors for LDB1-FLAG (Ldb1), poly-His-Ubiquitin-K48R (His-Ub) and AU1-RNF12 (RNF12) as indicated. His-containing proteins were isolated by chromatography and analyzed by gel electrophoresis as above. Closed arrows, migration appropriate for LDB1-FLAG, open arrows, more slowly migration form of LDB1-FLAG.