FIGURE 6.

Integrin-ILK-Akt signaling was also enhanced in GD3+ cells. A, phosphorylation of Akt during adhesion to CL type I in GD3+ cells (G5) and GD3− cells (V4) was examined. Cells were prepared as described in the legend to Fig. 3, and immunoblotting was performed using anti-phospho-Akt (Ser-473), anti-phospho-Akt (Thr-308), and anti-total Akt antibodies. B, band intensities were plotted after correction with those of total Akt. C, knockdown of ILK. To knockdown ILK, 4 kinds of anti-ILK siRNAs were analyzed. siRNA:si-ILK3 suppressed ILK levels most efficiently (70–80%), and was used hereafter. GL-2 (anti-firefly luciferase siRNA) was used as a control. D, ILK is involved in Akt phosphorylation. Effects of ILK knockdown on phosphorylation levels of Akt in GD3+ (a) and GD3− cells (b) during adhesion to CL type I were examined. After 48 h of transfection, cells were plated on pre-coated CL type I, and lysed for immunoblotting at incubation time 0 and 20 min. Definite reduction in Akt phosphorylation was detected in GD3+ cells. Bars indicate mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01. These were representative results among experiments repeated at least 3 times with similar results (C and D).