Analysis of translational regulation of STAT4 protein. (A) NKL cells treated without (-D) or with 50μM carmustine (Car) or 2μM etoposide (Eto) as described in Figure 3D were used for [35S] methionine and cysteine labeling (10 μCi/μL; Perkin Elmer). Dead cells were removed from the culture using Ficoll centrifugation, and the collected viable cells at 10 × 10⁶ cells/2 mL were subsequently pulsed for 15, 30, and 60 minutes with [35S] methionine/cysteine. The cell extract (1%) was separated by SDS-PAGE followed by exposure to X-ray film after drying on a Bio-Rad gel dryer. The molecular weight (MW) is accompanied on the side of the gel. (B) NKL cells treated as described in panel A were pulsed with [35S] methionine/cysteine for 24 hours. STAT4 and STAT3 proteins were immunoprecipitated from the total protein extracts using polyclonal anti-STAT4 and anti-STAT3 Abs, which were subjected to SDS-PAGE and then exposure to X-ray film after drying. A preimmunized rabbit serum (IgG) was used in immunoprecipitation as a negative background control. One percent of the total protein extracts from each sample was separated on the same gel as loading control (1% loading). The molecular weight (MW) at 75 kD is labeled.