FIGURE 5.

Identification of Cgi-58 regions involved in binding to A-Fabp. a, in solid phase assays, polystyrene plates were coated with purified A-Fabp and increasing amounts of purified Gst, Gst-Cgi-58, or N-terminally truncated Cgi-58 variants. Binding of proteins was detected using anti-Gst primary and Hrp-conjugated secondary antibody. Plates were developed using tetramethylbenzidine as substrate, and the absorbance was measured at 450/620 nm. b, SDS-PAGE of purified Gst, Gst-Cgi-58, and N-terminally truncated Cgi-58 variants after Coomassie Brilliant Blue staining. c, solid phase assay detecting the interaction between purified A-Fabp and His-tagged human Cgi-58, the naturally occurring mutant variants of human Cgi-58, and LacZ (control) contained in lysates of transfected COS-7 cells. d, Western blot analysis of respective cell lysates using anti-His primary and Cy3-conjugated secondary antibody. Statistical difference was determined as compared with Gst/LacZ control (*, p < 0.05; **, p < 0.01; ***, p < 0.001) and of 0.6/60 and 0.3/30 μg versus 0.1/10 μg of each lysate (§, p < 0.05; §§, p < 0.01; §§§, p < 0.001).