Fig. 3

Annexin A2 on the macrophage cell surface interacts with AcGal-1. a The different types of proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by Coomassie brilliant blue staining. The “pull-down protein” (red box) that may interact with AcGal-1 was obtained using His-Tag-based immunoprecipitation assay. b Immunofluorescence detection of cell surface Annexin A2 and colocalization with AcGal-1, which was abolished by treatment with the glycosylation inhibitor lactose. Macrophages were immunolabeled for Annexin A2 (revealed by FITC, green) and stained for AcGal-1 with (TRITC, red), and then examined using confocal microscopy. c, d Interaction of Annexin A2 and AcGal-1 were verified by IP, followed by western blot detection of Annexin A2 and AcGal-1. c Resin with affinity-purified His-antibody was incubated with AcGal-1-treated or untreated macrophage cell lysates. The Annexin A2 that interacted with His-AcGal-1 was pulled down along with His-mediated immunoprecipitation (IP) and revealed by immunoblot (IB) with the Annexin A2 antibody. AcGal-1 was blotted using the anti-His antibody. d Reverse co-IP confirmed the interaction between AcGal-1 and Annexin A2. AcGal-1-treated macrophage cell lysates were incubated with or without the resin bound by affinity-purified Annexin A2-antibody. AcGal-1 that interacted with Annexin A2 was pulled down along with Annexin A2-mediated IP and revealed by IB with the anti-His antibody. Annexin A2 was blotted using the Annexin A2 antibody. Scale-bars: b, 10 µm