FIGURE 3.
CN-mediated dephosphorylation of p-YB-1S102 decreases CCL5 expression in monocytes (THP-1 cells). Rat mesangial (rMCs) (A) or THP-1 cells (B) were transfected with a CN-GFP expression plasmid or control vector, and cell lysates were analyzed for p-YB-1S102 and total YB-1 content by immunoblot analyses. Densitometric analyses were performed on p-YB-1S102 bands with normalization against values determined for GAPDH. Relative band intensities are depicted in bar diagrams below. C, ChIP assays were performed in THP-1 cells transfected with a CN-GFP expression plasmid or control vector. Four hours after transfection, cells were incubated with PMA for 1 h, and ChIP was performed using a p-YB-1S102-specific antibody or unspecific IgG (negative control) with oligos within the CCL5 promoter region. The amount of included DNA was tested without preceding immunoprecipitation (input). CCL5 promoter activity (D), CCL5 mRNA (E), and CCL5 protein secretion (F) were determined in THP-1 cells transfected with a CN-GFP expression plasmid or control vector. The cellular YB-1 content was manipulated by transfection of THP-1 cells with YB-1-pSG5 expression plasmid (D). Cells were harvested 6 or 24 h after transfection and CCL5 mRNA (E) and secretion (F) was measured by TaqMan and ELISA technology, respectively, and normalized to control transfection. G, overexpression of CN was confirmed by TaqMan analysis of THP-1 cells transfected with the CN-GFP expression vector. H, Western blot analyses of cytoplasmic (CE) and nuclear proteins (NE) of THP-1 cells in the course of PMA-induced monocyte differentiation. Compartmental localization of CN was assessed using a specific polyclonal antibody. Separation of cell compartments and equal protein loading were ensured by GAPDH and CREB levels. I, nuclear protein extracts from THP-1 cells prior to incubation for 24 or 48 h with PMA or left untreated were prepared and complex formation with the antisense element of the CCL5 promoter was assessed. A strong high-mobility nucleoprotein complex appeared after 48 h of PMA-induced differentiation, which was not detected in the presence of a CN-specific polyclonal antibody (arrowhead) but with a p-YB-1S102-specific or nonspecific IgG-antibody as control. Experiments were performed in at least three independent experiments, each performed in triplicate. Data are expressed as mean values ± S.D. NE, nuclear extract.