Figure 4

Minimum expression of IL-2 mRNA in Tax2-transformed cells. Total RNA was isolated from indicated Tax-transformed CTLL-2 cells (lanes 3–8), or EL-4 T-cell line treated with (lane 2) or without (lane 1) 20 μg/ml phorbol myristate acetate and 1 μM ionomycin for 5 hours using RNAiso reagent, according to the manufacturer's instructions (Takara, Kyoto, Japan), and then total RNA (500 ng) was reverse transcribed using ExScript RT reagent kit (Takara). To quantify the amount of IL-2 RNA, a real-time polymerase chain reaction (PCR), based on SYBR green fluorescence, was performed using SYBR Premix Ex Taq polymerase and Takara real-time Thermal Cycler Dice (Takara). The following primers were used to specifically amplify respective genes: mouse IL-2 gene, 5'-GGAGCAGCTGTTGATGGACCTAC-3' and 5'-AATCCAGAACATGCCGCAGAG-3', mouse glyceraldehyde-3-phosphate dehydrogenase gene used as a control, 5'-TGTGTCCGTCGTGGATCTGA-3' and 5'-TTGCTGTTGAAGTCGCAGGAG-3'.