Thiol depletion of NK3.3 cells reduced RB expression and greatly reduced or abrogated phosphorylation of RB in response to IL-2. (A) Western blot analysis of RB protein in NK3.3 cells cultured with rIL-2 (200 U/mL) for 0 to 24 hours in CYS(+) medium or CYS(−) medium. Phosphorylated RB migrates slower than nonphosphorylated RB.32 33 (B) Addition of thiol-related reducing reagents, GSH, 2-ME, or NAC to CYS(−) medium restored RB phosphorylation and expression in response to IL-2 (Western blot) and [³H]-TdR incorporation (bar graph) in a dose-dependent manner. Controls included cells without IL-2 in CYS(−) medium (IL-2[−]), and NK3.3 cells cultured in CYS(−) medium with IL-2 (200 U/mL) (Cystine [0]). (C) RB phosphorylation is induced by IL-2 in freshly isolated human NK cells. Immediately after isolation (time 0), cells were cultured with IL-2 (200 U/mL) in CYS(−) or CYS(+) medium. Each data set is representative of two independent experiments.