Figure 4.

Gal3 negatively regulates complement receptor 3 downstream Syk-mediated neutrophil reactive oxygen species (ROS) response to Candida. Gal3^+/+ (WT), gal3^−/−, and Itgam^−/− neutrophils were stimulated with opsonized Candida (Ops. Ca). (A) Cell lysates were collected at different time points after stimulation and subjected to Western blot analysis with anti-p-Syk antibody. β-actin was used as a loading control. (B) WT cells were pretreated with 2, 10, and 50 µM of Syk inhibitor before stimulation. The levels of ROS produced by neutrophils at 20 min after stimulation were analyzed by flow cytometry. Relative ROS production was calculated by dividing mean fluorescence intensity of oxidized DCF fluorescence in Candida-stimulated cells by that in untreated cells. n = 6 as pooled from two independent experiments. (C) Cell lysates were collected at different time points after stimulation and subjected to Western blot analysis with anti-p-Syk, -Syk, or -p-p40^phox antibodies. β-actin was used as a loading control. Relative intensity of p-Syk (n = 5) and p-p40^phox (n = 2) at different time points are shown in the right panel. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.005, as analyzed by Mann–Whitney test. (D) Whole cell lysates (WCL) were immunoprecipitated with anti-gal3 (IP: Gal3, upper panel) and anti-Syk (IP: Syk, lower panel) antibodies. Precipitates were subjected to Western blot analysis with anti-Syk and anti-gal3 antibodies. Experiments were performed three times. Data from one representative experiment are shown. (E) Confocal microscopy image of gal3 (red) and Syk (green) in neutrophils after stimulation by Candida. The intensity of different fluorochromes along the white arrow in the merged image is shown in the histogram on the right. See also Figure S1 in Supplementary Material.