Effect of rapamycin on PMA/CD28-induced IL-2 mRNA.

(A) Total RNAs (10 μg) from peripheral blood T cells treated with PMA plus αCD28 in the presence (right panel) or absence (left panel) of rapamycin for various periods of times were analyzed by Northern blot analysis (upper panel). A polymerase chain reaction–amplified fragment corresponding to a portion of IL-2 cDNA was used as a probe. The same membrane was stripped and reprobed with an 18s ribosomal RNA probe (lower panel). (B) Graphical representation of the IL-2 mRNA levels quantitated and normalized with 18S ribosomal RNA. (C) Northern blot analysis using total RNAs (10 μg) from T cells stimulated with either medium alone, PMA/αCD28, or PMA/ionomycin in the presence or absence of rapamycin for 18 hours. (D) Peripheral blood T cells were stimulated for 18 hours in the presence or absence of rapamycin. Actinomycin D was then added, and the cultures were incubated further for various periods of times. After each point cells were harvested; total RNAs were isolated and analyzed by Northern blot analysis.