Fig. 2

Binding of EDP to the ERC decreases the sialylation level of β₂ integrin through NEU1 in human monocytes. a, b Left panels: SNA and MALII pull down of crude membrane preparations of THP-1 cells 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 β₂ integrin in α-2,6 (SNA) or α-2,3 (MALII) recovered after lectin pull down was evaluated by Western blot using a mouse monoclonal anti-β₂ integrin antibody. The image is representative of 4 independent experiments. Right panels: quantification of α-2,6 and or α-2,3 sialylation levels of β₂ integrin (pull down/lysate ratio) by densitometry analysis, and normalized to the basal condition (without κE, w/o). Results are expressed as mean ± SEM of 4 independent experiments and statistical analysis was performed by Student’s t-test (*p < 0.05; **, p < 0.01; ns, non-significant). c Top, western blot on the biotinylated and cell lysate fractions of THP-1 cells stimulated, or not, with κE (50 µg/mL, 1 h), and probed with a mouse monoclonal anti-β₂ integrin antibody. A representative pattern is shown. The NS lane corresponds to non-biotinylated cells used for estimation of non-specific binding of β₂ integrin to streptavidin beads. Bottom, quantification of the relative expression of β₂ integrin in the biotinylated fraction by densitometry analysis. Relative expression was calculated as amount of β₂ integrin 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