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. Author manuscript; available in PMC: 2010 Nov 1.
Published in final edited form as: Biochem J. 2009 Oct 12;423(3):315–321. doi: 10.1042/BJ20091170

Figure 2. MIF is a target for ITC modification.

Figure 2

A: HeLa cells were seeded in 12 well plates and transfected with a pool of siRNAs directed against MIF or the control non-targeting siRNA pool (100pmoles/well) as indicated. Thirty minutes prior to harvest, indicated wells were pretreated with 250μM PEITC (pre tx). Cells were lysed and labeled with Bio-ITC as described in Figure 1. Effective depletion of MIF from the lysates (lower panel) was accompanied by loss of Bio-ITC reactivity of the Bio-ITC target protein (upper panel). Panel B: CBD-tagged MIF was expressed in HeLa cells, pretreated with PEITC or not, and cell lysates labeled with Bio-ITC as above. CBD tagged proteins were purified on chitin beads, and Bio-ITC labeling detected by immunoblot. CBD-tagged MIF was efficiently labeled by Bio-ITC, and this label was completely inhibited by pretreatment of the cells with PEITC.