Figure 4.

TIMP-1 promotes the activation of serine/threonine kinases in THP-1 monocytic cells. Cells were incubated under serum-free conditions in the absence (untreated or 0) and presence of recombinant TIMP-1 (100 ng/mL) for different time intervals, as indicated. (a) Total-protein cell lysates were obtained and analyzed using a membrane-based phosphokinase array, detecting 46 different kinase phosphorylation sites (seven of the phosphorylated substrates are indicated; the respective phosphorylated amino acids in brackets). (b) Densitometric quantification of the spots in (a) and representation as a heatmap. The quantification is based on mean values from duplicate determinations in percent to untreated control cells (100%). The heatmap shown provides an overview of the changes in phosphorylation of all 46 measured kinase substrates/sites at all three determined time points. (c) Selection of the heatmap in (b) with a focus on those substrates/sites with the most-substantial alterations. (d) Representation of the densitometric quantification according to the selection of substrates/sites depicted in (c) as a bar diagram. The dotted red line indicates the baseline value of the control. (e) Western blot analysis of cell lysates using antibodies against phosphorylated AKT (p-AKT), total Akt (AKT), and β-actin as the loading control.