Jak3is a primary response gene for IL-6. (A) M1 cells were treated with medium alone (lanes 1-6), medium containing IL-6 in the absence or presence of cycloheximide (CHX, 10 μg/mL; lanes 7-18), or medium containing cycloheximide alone (lanes 19-24). Total cellular RNA was extracted at 0, 2, 4, 6, 8, and 24 hours following cytokine treatment and 20 μg RNA was used for Northern analysis. The level of expression of Jak3 was determined by hybridization to a ³²P-labeled probe (upper panel). To control for the amount of RNA loaded, the membrane was stripped and reprobed with a ³²P-labeled G3PDH probe (lower panel). (B) Cells were plated in IL-6-free medium (lanes 1-4) or in medium containing IL-6 (lanes 5-8) for 24 hours. Actinomycin D (Act D) was then added to the cultures and incubation was further continued for the indicated hours. RNA (20 μg) from each sample was subjected to Northern blot analysis. (C) M1 cells were stimulated with IL-6 for the indicated hours. Cells were pelleted and the labeled RNA was subjected to nuclear run-on assays as described in “Materials and methods.”