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. 2010 Oct 14;286(1):607–619. doi: 10.1074/jbc.M110.153122

FIGURE 2.

FIGURE 2.

CHIP binds to an Arg-Lys-Ser-rich motif in the Mf2 domain of IRF-1. A, biotin-tagged IRF-1 peptides (60 pmol) spanning the entire length of the protein were immobilized on streptavidin-coated microtiter wells and incubated with His-CHIP (25 ng); binding was detected using anti-His mAb. CHIP binding to the IRF-1 peptides, expressed as relative light units (RLU) is shown. The results are representative of four separate experiments. B, His-CHIP (100 ng) was preincubated with a titration of the indicated IRF-1 peptides and added to immobilized GST-IRF-1 (100 ng). CHIP binding was detected and expressed as above. A C-terminal IRF-1 peptide (sequence MDATWLDSLLTPVRLPSIQA) that did not bind CHIP (see Fig. 2A) was used as a control. C, His-CHIP (100 ng) was immobilized on microtiter wells and incubated with a titration (0–12.5 ng) of GST-IRF-1 WT, GST-IRF-1 Δ106–140, or GST alone. Binding was detected using an anti-GST monoclonal antibody. The results are representative of two independent experiments. Right, normalization of protein levels using anti-GST antibody.