l-Arg starvation impairs expression of cyclin D3 and cdk4 through transcriptional, posttranscriptional, and translational mechanisms. (A) RNA (5 μg) from nonactivated human T cells or T cells activated with anti-CD3 plus anti-CD28 and cultured in the presence or the absence of l-Arg was tested for cyclin D3, cdk4, and cdk6 RNA expression by Northern blot. (B) Nuclei obtained from activated T cells cultured in the presence and the absence of l-Arg were tested for cyclin D3, cdk4, and cdk6 transcriptional rate by run-on analysis as described in “Materials and methods.” (C) RNA stability was also tested in activated T cells cultured in the presence and the absence of l-Arg for 12 hours, after which Act D (5 μg/mL) was added and RNA collected after 3, 6, 12, 24, and 36 hours. RNA expression was then tested by Northern blot. (D) Nonactivated (NS, lane 1) and activated (lanes 2-3) T cells were cultured in the presence or the absence of l-Arg for 48 hours, after which cells were washed and pulsed with 250 μCi (9.25 MBq) [³⁵S] methionine for 3 hours. Lysates were immunoprecipitated using anti–cyclin D3 as described in “Materials and methods.”