Figure 6.

Binding of CvGal1 and CvGal2 to oyster hemocytes. (A) Upon hemocyte attachment and spreading, CvGal1 is translocated to the periphery and secreted. Western blotting of unattached hemocytes, plasma, attached-spread hemocytes, and supernatant. (B) Immunofluorescence staining with anti-CvGal1 and DAPI staining of attached-spread hemocytes with (+) or without (–) Triton X treatment, showing the presence of CvGal1 in the cytoplasm, and on the external surface of the hemocyte plasma membrane, respectively. Scale bar, 10 μm. (C) Binding of rCvGal1 (100 μg/ml) to hemocytes in the presence of PSM (0–300 μg/ml), whereby the control (Ctrl) is sample without exogenous rCvGal1 and inhibitor. (D) Binding of rCvGal1 to unattached hemocytes with α-N-acetylgalactosaminidase treatment (GalNAc'ase) or no treatment (Untreated) were measured by flow cytometry, whereby a sample without rCvGal1 was the control (Ctrl). Data show mean fluorescence intensity (MFI) ± S.E. of each sample. (E) Fixed hemocytes were stained with dilutions of anti-blood group A antibody (red, 1:2000; blue, 1:500; yellow, 1:100) or buffer only (black) in flow cytometry analysis. (F) Fixed cells were preincubated with anti-blood group A antibody (1:100), and the binding of rCvGal1 (100 μg/ml) was measured by flow cytofluorometry; the control (Ctrl) was recorded in the absence of rCvGal1. *Indicates significant difference (p < 0.05) between samples from One-Way ANOVA analysis [Adapted from Tasumi and Vasta (2007) and Feng et al. (2013) with permission from the American Association of Immunologists and the American Society for Biochemistry and Molecular Biology].