Galectin-3 Plays an Important Role in BMP7-Induced Cementoblastic Differentiation of Human Periodontal Ligament Cells by Interacting with Extracellular Components

Figure 3

The antigenic molecule recognized by the anti-CM3 antibody turns out to be galectin-3. (a) Identification of the antigenic molecule recognized by the anti-CM3 antibody. (A) Detection of the CM3 antigenic molecule. The antigenic molecule labeled with biotin was detected by HRP-conjugated streptavidin (left panel) and by staining with Coomassie Blue (right panel). Lane 1, protein G beads from preclearing; lanes 2 and 3, immunoprecipitates with anti-CM3 antibody; lane 4, protein size marker. The antigenic molecule was indicated by an arrow. (B) Tandem mass spectrometric data of anti-CM3 immunoprecipitates. (b, c) Galectin-3 protein is recognized by the anti-CM3 antibody. Ectopically expressed galectin-3 containing FLAG tag and endogenous galectin-3 were detected by the anti-CM3 antibody. (b) Anti-FLAG immunoprecipitates (α-FLAG IP) were recognized by both anti-CM3 and anti-galectin-3 antibodies. Lane 1, vector only; lane 2, cDNA transfection. (c) Anti-CM3 antibody has cross-reactivity with commercially available anti-galectin-3 antibody. Anti-CM3 immunoprecipitates (α-CM3 IP) were recognized by anti-galectin-3 antibody (α-Gal3 IP) and vice versa in BMP7-induced human cementoblasts (A) and the mouse cementoblast cell line OCCM-30 (B). Lanes 1 and 3, protein amounts in cell extract; lanes 2 and 4, immunobinding in immunoprecipitates. The arrowheads indicate the light chain of IgG. (d) Epitope mapping of galectin-3 recognized by the anti-CM3 antibody. (A) The schematic presentation of galectin-3 domain constructs. All constructs tagged with FLAG (circle) were expressed in HeLa cells and were detected by anti-FLAG (B) and anti-CM3 (C) antibodies. The arrowhead in (C) indicates endogenous galectin-3 expressing in HeLa cells.

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