FIG. 6.
TGF-β-mediated growth inhibition of αCD3-stimulated splenocytes is associated with a decrease in G1 Cdk activity and cytokine expression. (A) Splenocytes were harvested from wild-type and knockout mice and cultured with αCD3 in the presence or absence of TGF-β. Cell lysates were prepared and subjected to Western blotting for cyclin E, Cdk2, p27, and Rb (lower panels). In addition, Cdk2 kinase activity was assayed by immunoprecipitation of Cdk2 and evaluation of its ability to phosphorylate the exogenous substrate, histone H1 (top panel). (B) Cytokine production was assayed on splenocytes from wild-type and Smad3 null mice treated as for panel A, using an RNase protection assay. The identity of each band is indicated on the right. L32 and GAPDH are controls for mRNA quantity and quality. (C) The intensity of the cytokine RPA bands in panel B was determined by densitometry. Plotted are the relative intensities of each band, with wild-type levels of each cytokine set at 100%.