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. 2008 May 5;28(13):4261–4274. doi: 10.1128/MCB.02252-07

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FIG. 5. — Physical interactions between CRP-1, HIM-6, and CDC-48. (A) Binary interactions between CRP-1 and either His₆-HIM-6 or His₆-CDC-48. The purified proteins were pulled-down using Ni-NTA-agarose beads, and the presence of GST-CRP-1 was revealed by immunoblotting (Ib) using anti-GST antibodies. (B) Binary interactions between His₆-CDC-48.1 and Strep-tagged HIM-6 or His₆-HIM-6 and Strep-tagged CDC-48.1 following purification of the complex by Ni-NTA-agarose beads and analysis by immunoblotting using anti-Strep tag antibodies. (C) Ternary complex including CRP-1, HIM-6, and CDC-48.1. Increasing concentrations of His₆-CDC-48.1 were added to an equimolar mixture of GST-CRP-1 and Strep-tagged HIM-6. Following 1 h of incubation on ice, the complex was pulled-down using glutathione-Sepharose beads and the presence of HIM-6 in the complex was evaluated by immunoblotting using anti-Strep tag antibodies.