Fig. 4

Panel (A) NP-40 detergent extracts prepared from RAW 264.7 cells (2 × 10⁵ cell equivalents/lane) were subjected to immunoprecipitation with either isotype control rat IgG2a (Lane 1) or anti-galectin-3 mAb M3/38 (Lane 2). After SDS-PAGE through a 12% gel and electrotransfer onto nitrocellulose, filters were subjected to ligand blotting using ¹²⁵I-AGE-BSA. (B) Purified 18-kD carboxyl-terminal domain of recombinant murine galectin-3 (3 µg/lane) was subjected to ligand blot analysis using ¹²⁵I-AGE-BSA in the presence (Lane 2), or absence (Lane 1) of 50-fold unlabeled AGE-BSA. (C) Filters were either stained with Amido black or probed with ¹²⁵I-AGE-BSA. Protein stain of the filters (1, 2) and autoradiography of the ligand blots (3, 4) are shown. (D) Quantitative analysis of ¹²⁵I-AGE-BSA binding to either intact galectin-3 or its 18-kD C-terminal peptide. Data from Panel C are expressed in phosphorimage units (PI)/mg protein.