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. 2016 Jun 28;6:28706. doi: 10.1038/srep28706

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Copyright © 2016, Macmillan Publishers Limited

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CDH23-C isoforms are able to bind CKK and harmonin simultaneously. GST, GST-tagged CDH23-C1 and CDH23-C2 proteins were immobilized on glutathione-agarose and incubated with equal amounts of cell lysates containing CKK domains with or without harmonin (uninduced). GST-agarose associated proteins were eluted with 100 mM L-glutathione and separated on a 12% SDS-PAGE gel. GST, GST-tagged proteins, and harmonin were visualized by Ponceau S at the top, while CKK1 (panel A) and CKK2 (panel B) were identified using anti-Marshalin (bottom). Both CDH23-C1 and CDH23-C2 pulled down CKK1 and 2 in the presence or absence of harmonin, while GST alone did not. In panel (C) CKK1 and harmonin were first purified separately. Similar amounts of CDH23-C1-GST bait and GST were mixed with 1 μM of CKK1 and harmonin at different molar concentrations, including CKK1: harmonin ratios of 1:0, 1:1, 1:2, and 1:4. Molecular weights (in kD) are labeled with numbers. The excess unbound CKK1 and harmonin were visualized by Coomassie Blue.