Figure 3.

(a) Northern analysis for IFN-γ and IL-4 in IL-12-pretreated Th0 clones. T-cell clones with or without IL-12 pretreatment were stimulated with immobilized anti-CD3 monoclonal antibodies. After 4 hr of stimulation the cells were harvested, total RNA was prepared and Northern analysis performed. The blots were sequentially probed for IFN-γ, IL-4 and β-actin. The autoradiograms were analysed and quantified using a Sigma Gel Program. The densitometric numbers obtained by the above analysis is indicated above each band of the autoradiogram. Lane 1, IL-12 untreated cells; lane-2, cells pretreated with IL-12 for 48 hr. The percentage increase in IFN-γ message was calculated as (B/F−A/E)/A/E×100. The percentage increase in IL-4 message was calculated as (D/F−C/E)/C/E×100. (b) Determination of mRNA stability. Three Th0 clones were pretreated with IL-12 for 48 hr or left untreated. The cells were then stimulated and 2 hr later actinomycin D was added at 10 μg/ml. Total RNA was isolated at 75-min intervals and subjected to Northern analysis. Data obtained from one such clone are depicted in this figure. Lanes 1–6 represent different time-points at which IL-12 untreated cells were harvested, and lanes 7–12 represent different time-points at which IL-12 pretreated cells were harvested. (c) Transcription of IFN-γ and IL-4 message in IL-12 pretreated clone Th0-1. This was determined by the thiouridine method. Lane 1, eluted fraction of IL-12 untreated cells; lane 2, flow-through of IL-12 untreated cells; lane 3, eluted fraction of IL-12 pretreated cells; lane 4, flow-through of IL-12 pretreated cells.