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. Author manuscript; available in PMC: 2010 Dec 1.
Published in final edited form as: Cell. 2010 Jun 11;141(6):1006–1017. doi: 10.1016/j.cell.2010.04.040

Figure 1. 2,5-DHBA is the Iron-Binding Moiety of the 24p3-Associated Mammalian Siderophore.

Figure 1

(A) Immunoblot analysis showing 24p3 in the CM of FL5.12 cells expressing either vector or Ec-24p3 in the presence or absence of ponasterone A.

(B) Iron binding assay. 24p3 was immunoprecipitated from the CM of cells described in (A) and analyzed for the presence of 55Fe. CPM, counts per minute. Error bars indicate SD.

(C) (Top) GC-MS analysis on the TMS-derivatized, small molecule flow-through fraction eluted from immunopurified 24p3. (Bottom) Matching spectrum from the NIST database.

(D) GC-MS analysis on the eluted sample from immunopurified 24p3 and a set of DHBA standards.

(E) Chemical structures of 2,3-DHBA and 2,5-DHBA.

(F) Tryptophan fluorescence quenching assay. 2,3-DHBA (left) or 2,5-DHBA (right) was added to apo-24p3 at increasing concentrations, and tryptophan fluorescence was monitored. Error bars indicate SD.

(G) Iron binding assay. Purified apo-24p3 was incubated with 55FeCl3 and increasing concentrations of either 2,3-DHBA, 2,5-DHBA, iron-free enterobactin or desferrichrome, and radioactivity was measured in the 24p3 immunoprecipitate. Error bars indicate SD. See also Figure S1.