The IFN-γ 3′ UTR mediates regulation of mRNA stability by the p38 pathway. (A) HeLa-TO cells were transfected with 100 ng of pTetBBB-IFN-γ and 100 ng of pCDNA3 or pCDNA3-MKK6E (which expresses a constitutively active mutant of MKK6). Total DNA was made up to 1 μg by addition of carrier (pBluescript; Stratagene, La Jolla, CA). After 24 hours, cells were treated with 1 μM SB203580 (SB) or vehicle (0.1% DMSO) for 5 minutes prior to addition of tetracycline (final concentration 100 ng/mL). Cells were harvested at the intervals shown and ribonuclease protection assays performed to quantify β-globin-IFN-γ (reporter) and GAPDH (loading control) mRNAs. This experiment was performed 3 times, reporter mRNA levels were normalized against GAPDH, and mean outcomes plotted on a semilogarithmic graph (bottom). Error bars indicate SEM. (B) HeLa-TO cells were transfected with 100 ng of pTetBBB-IFN-γ and 800 ng of pEF or pEF-MK2ca (which expresses a constitutively active mutant of MK2). Total DNA was made up to 1 μg by addition of carrier (pBluescript). After 24 hours, tetracycline was added and cells were processed as described above. Mean outcomes of 3 independent experiments were plotted as described for panel A. (C) HeLa-TO cells were transfected with 100 ng of pTetBBB-IFN-γ, 100 ng of pCDNA3 or pCDNA-MKK6E, and 800 ng of pEF or pEF-MK2dn (which expresses a dominant-negative form of MK2). After 24 hours, tetracycline was added, cells were processed, and mean outcomes of 3 independent experiments were plotted as described for panel A.