Pierce protein g plus agarose

To analyze RNAs binding to PCBP4 protein, RIP Assay was performed using a RiboCluster Profiler^TM RIP Assay kit (Medical & Biological Laboratories Co., Ltd., Nagoya City, Aichi, Japan) following the protocols of the manufacturer. Briefly, IMC-3PCBP4 cells (1 × 10⁷) were treated with cisplatin (1 μg/ml) for 24 h. Lysis of the cells was performed after washing. The protein G beads (Pierce^TM Protein G Plus Agarose, Thermo Fisher Scientific, Waltham, MA, USA) were pre-incubated with anti-PCBP4 antibody (Santa Cruz Biotechnology Inc.) or normal IgG (Medical & Biological Laboratories Co., Ltd.). Then RNA-protein immunocomplexes were precipitated using the pre-incubated protein G beads. The RNAs were isolated and subjected to RT-PCR of Cdc25A, Cdc25B, and Cdc25C. The previously described primers for real-time PCR were used.

The expression protocol used here was based on the Expi293^TM Expression System (ThermoFisher #A14635). Expi293 suspension cells were seeded at a final density of 2.5 ×10⁶ cells/mL in a 6-well plate. The next day cells were diluted to a final density of 3 ×10⁶ cells/mL and a total of 1.0 μg/mL of plasmid DNA was transfected using ExpiFectamine^TM 293 Transfection Kit. Transfected cells were then incubated on an orbital shaker at 37 °C and 8% CO₂ overnight and ExpiFectamine 293 Transfection Enhancer 1 and 2 were added to the cells. Secreted IgG was harvested 6 days post-transfection and the supernatant was incubated with Pierce^TM Protein G Plus Agarose (ThermoScientific #22851) resin at room temperature for 1 h. After washing with 10 column volumes of PBS, the IgG was eluted with 2 column volumes of 100 mM glycine, pH 2.5 and neutralized by 1 M Tris, pH 8.5. The eluted fractions were determined by SDS-PAGE gel and fractions containing pure IgG were collected and dialyzed against PBS buffer.