Figure 2.

(See previous page). CD5L inhibits RELA phosphorylation and subsequent nuclear translocation. (Left) PMA-differentiated THP1-Vector (white boxes) and THP1-HsCD5L (black boxes) MФ and (Right) PB monocytes incubated for 24 h with 1 μg/ml albumin (ALB) as control protein (white-dotted boxes) or 1 μg/ml r-HsCD5L (black-dotted boxes) were (A) stimulated for the indicated times with 1 μg/ml of Pam3CSK4 or LPS, lysed, and probed in western blot with an antibody specific to phosphorylated RELA (Ser536) and TUBB2A. Upper panel: Western blot images of a single experiment. Lower panel: mean of protein signal intensities ± SEM of 3 independent experiments (for THP1 MФ) or PB monocytes obtained from 3 healthy donors. Fold increase is relative to THP1-Vector MФ or ALB-PB monocytes at time 0 after normalization with the loading control protein TUBB2A. (B) Cells were stimulated with 1 μg/ml Pam3CSK4 (Pam3) or LPS for 1 h. Cells were fixed, and the RELA subunit of the NFKB complex was stained with a specific antibody (green) and nuclei with Hoechst dye (blue). Upper panel: representative confocal microscopy images of THP1 MФ (left) and PB monocytes (right). Lower panel: mean nuclear vs. cytoplasmic RELA fluorescence intensity ratio values ± SEM in more than 200 cells scored in random fields, from 3 independent experiments (for THP1 MФ) or PB monocytes obtained from 3 healthy donors *P ≤ 0.05; ***P ≤ 0.001, one-way ANOVA. M are untreated cells (left in culture medium).