FIG. 7.

RPA. (A) Induction of cytokine mRNAs by K1. BJAB cells (10⁷) were electroporated with 20 mg of pcDEF/K1 WT, and live cells were separated by Ficoll-Hypaque centrifugation at 0, 6, 12, 24, and 36 h postelectroporation. Total RNA was isolated by phenol-chloroform extraction, and contaminated DNA was removed by DNase I treatment. Two milligrams of total RNA was then subjected to RPA using an RPA kit (BD PharMingen, San Diego, CA) according to the manufacturer's recommendations. ³²P-labeled probes before and after RPA were included at the left and right sides of the gel, respectively, as controls. Reverse transcription-PCR was performed to detect K1 and actin expression (bottom). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Cytokine mRNA production by WT K1 but not by TYF mutant K1. BJAB cells (10 × 10⁶) were electroporated with 20 mg of pcDEF, pcDEF/K1, or pcDEF/K1TYF. Live cells were harvested and separated by Ficoll-Hypaque centrifugation at 18 h. RPA was carried out as described above. ³²P-labeled probes before RPA were included at the left side of the gel as controls. Reverse transcription-PCR was performed to detect WT K1, TYF mutant K1, and actin expression (bottom). V, vector. (C) Kinetics of cytokine mRNA production induced by K1. Signal intensities are shown as kinetics. The values shown were obtained by subtraction of the background signal intensity.