Jak3 is a primary response gene for G-CSF.
(A) 32Dcl3 cells were treated with medium alone (lanes 1-6) and medium containing G-CSF in the absence or presence of cycloheximide (10 μg/mL; lanes 7-18). 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 Jak3 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-3–free medium (lanes 1-4) or in medium containing IL-3 (lanes 5-8) or G-CSF (lanes 9-12) for 12 hours. Actinomycin 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) 32Dcl3 cells were stimulated with G-CSF for 2 hours. Cells were pelleted and the labeled RNA was subjected to nuclear run on assays as described in “Materials and methods.” (D) Expression of Jak3 protein in G-CSF–stimulated 32Dcl3 cells. Cells were harvested at the indicated time (days) after exposure to G-CSF and 1.2 mg of the cell lysates was used for immunoprecipitation. The level of Jak3 protein was determined by probing the membrane with an anti-Jak3 antibody (upper panel) and presence of tyrosine phosphorylated proteins was detected by probing with the antiphosphotyrosine antibody, 4G10 (UBI, lower panel).
