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. Author manuscript; available in PMC: 2014 Mar 1.
Published in final edited form as: Mutat Res. 2012 Dec 19;0:89–96. doi: 10.1016/j.mrfmmm.2012.12.002

Figure 3. DNA topoisomerase I interacts in vitro with the C-terminus of BLM.

Figure 3

In vitro transcription/translation (IVTT) was performed using the TNT Rabbit Reticulocyte Lysate kit (Promega) using pET24D-BLM-N, pET24D-BLM-H and pET24D-BLM-C. The resultant 35S-methionine-labeled input BLM fragments are shown on the left. N-, H- and C-fragments were used in co-immunoprecipitation with purified DNA topoisomerase I protein (Topogen) and α DNA topoisomerase I. Following capture with Protein G Dynabeads (Invitrogen), immunoprecipitated proteins were separated using 8% SDS-PAGE; western blotting identified DNA topoisomerase I; autoradiography identified BLM fragments. Negative control immunoprecipitations were carried out by omitting DNA topoisomerase I to control for non-specific binding of BLM fragments to α DNA topoisomerase I or to the beads.