Total RNA was extracted using PrepEase RNA spin kit (USB, Cleveland, OH). We used Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN) for cDNA synthesis. Quantitative PCR was performed using predesigned Taqman gene expression assays (Life Technologies, Carlsbad, CA) and the 7900HT or ViiA 7 real-time PCR systems (Applied Biosystems/Life Technologies). TBP was used as a housekeeping control gene. mRNA stability was assessed by performing quantitative PCR after actinomycin D treatment. For immunoblotting, antibodies recognizing RBM47 (HPA006347; Sigma, St. Luis, MO), α-tubulin (11H10; Cell Signaling, Danvers, MA) and ACTB (Sigma) were utilized. Secondary antibodies were HRP (Pierce, Rockford, IL) or fluorescence (LiCor, Lincoln, NE) conjugated. Immunostaining for RBM47 (HPA006347; Sigma) was performed according to standard protocols in the MSKCC Molecular Cytology Core Facility on paraffin embedded tissue blocks. Secreted protein was detected by ELISA (R&D, Minneapolis, MN).
Vanharanta S., Marney C.B., Shu W., Valiente M., Zou Y., Mele A., Darnell R.B, & Massagué J. (2014). Loss of the multifunctional RNA-binding protein RBM47 as a source of selectable metastatic traits in breast cancer. eLife, 3, e02734.
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