Rnase protection assay.
Total RNA was isolated from CD19+ MACS-purified B cells using the Rneasy midi purification kit with Dnase treatment, according to the manufacturer's instructions (Qiagen). The mCK-1 probe template set (BD Biosciences) was labeled using the In Vitro Transcription Kit according to the manufacturer's instructions (BD Biosciences). Briefly, 50 ng of mCK-1 probe set was labeled with [α-32P]UTP using T7 RNA polymerase and purified using Sephadex G-50 columns (NucAway Spin column, Ambion). The labeled probe was quantitated using a scintillation counter, and 6 × 105 cherenkov cpm was used to hybridize to 4 μg of total RNA using the RPA kit (BD Biosciences). Samples were denatured at 90 °C and hybridized at 56 °C overnight. Single-stranded RNA was digested with a mixture of Rnase A and T1, and precipitated using isopropanol. Protected probes were resolved on a denaturing 4.75% acrylamide gel, dried, and imaged using a phosphorImager (Molecular Dynamics). Densitometry was performed using the ImageQuant TL v2005 software (GE Healthcare).