Fig. 5

Binding of EDP to the ERC decreases the sialylation level of ICAM-1 through NEU1 in human endothelial cells. a Left panel: SNA pull down of crude membrane preparations of PMA-pre-stimulated HUVEC incubated, or not, with κE (50 µg/mL), V14 + κE (molar ratio 2:1) or V14 peptide alone for 1 h at 37 °C. For each condition, equal amount of proteins was used. The amount of sialylated ICAM-1 recovered after lectin pull down was evaluated by Western blot using a mouse monoclonal anti-ICAM-1 antibody. The image is representative of 3 independent experiments. Right panel: quantification of α-2,6 sialylation level of ICAM-1 (pull down/lysate ratio) by densitometry analysis, and normalized to the basal condition (without κE, w/o). Results are expressed as mean ± SEM of 3 independent experiments and statistical analysis was performed by Student’s t-test (*p < 0.05; ns, non-significant). b Top, western blot on the biotinylated and cell lysate fractions of PMA-pre-stimulated HUVEC incubated, or not, with κE (50 µg/mL, 1 h), and probed with a mouse monoclonal anti-ICAM-1 antibody. A representative pattern is shown. The NS lane corresponds to non-biotinylated cells used for estimation of non-specific binding of ICAM-1 to streptavidin beads. Bottom, quantification of the relative expression of ICAM-1 in the biotinylated fraction by densitometry analysis. Relative expression was calculated as amount of ICAM-1 recovered in the biotinylated fraction over expression level in cell lysate and normalized to the basal condition (w/o). Results are expressed as mean ± SEM of 3 independent experiments