FIGURE 5.

p18 is stable and associated preferentially with CDK6 in T cells. A, Stable levels of p18 during T cell activation. Total levels of CDK6, CDK4, p27, and p18 in freshly isolated LN T cells (lane 1) or cultured for 36h (5 × 10⁶/ml) without (lane 2) and with (lane 3) CD3/CD28 activation were determined by IP-Western blot analysis with specific Ab. B, Preferential association of p18 with CDK6 in activated T cells. Coimmunoprecipitation of p18 with CDK4 (lane 1) and CDK6 (lane 2) in CD3/CD28-activated T cells (from Fig. 5A) was performed. The IP pellets were immunoblotted with anti-CDK4 (top left), anti-CDK6 (top right), or anti-p18 Ab (bottom panels). C, Preferential association of CDK6 and p18 in primary T cells stimulated with anti-CD3 and/or anti-CD28 mAbs. LN T cells were cultured in the absence (lane 1) or presence of anti-CD3 (lane 2), anti-CD28 (lane 3), and both anti-CD3 and anti-CD28 (lane 4). The cells were harvested at 36 h post activation and analyzed by IP (with Abs labeled on top of the panels) and Western blot (labeled on the left and right) assays. Densitometry results (mean density) are presented. ■, Level of p18 associated with CDK6 (bottom right panel); □, p18 associated with CDK4 (bottom left panel). The band below the CDK6 band in the CDK6 IP was not related to CDK4, because immunoblotting of the anti-CDK6 IP with the anti-CDK4 Ab did not detect this band.