Protein g agarose

RD cells (2.4×10⁶/10-cm dish) were seeded 24 h prior to EV71 infection at a MOI of 40. Cells were lysed in 1 ml of IP-lysis buffer (25 mM Tris-HCl pH 7.6, 300 mM NaCl, 0.5% CA630, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, and 1× proteinase inhibitor) at 4°C for 30 min and then treated with 10 µg/ml RNase A at 30°C for 1 h. The cell extracts were pre-cleared by incubation at 4°C for 1 h with protein G-agarose (GE Healthcare) and centrifuged to remove non-specific complexes. The lysate was then added to 10 µg/ml EV71 3D^pol antibody or Prp8 antibody (Abcam) at 4°C for 2 h and 100 µl of protein G-agarose at 4°C for 12 h. The co-precipitated proteins were collected by centrifugation, followed by washing 6 times with IP buffer (25 mM Tris-HCl pH 7.6, 200 mM NaCl, 0.1% CA630, 6% glycerol, 1 mM EDTA, 0.5 mM DTT, and 1× proteinase inhibitor). The precipitated proteins were separated by 8% SDS-PAGE; subsequently, these immune complexes were detected using anti-Prp8 (diluted 1∶5000; Abcam), Brr2 (diluted 1∶5000; Abcam), Snu114 (diluted 1∶5000; Abcam), Prp6 (diluted 1∶5000; Abcam), SNRNP40 (diluted 1∶5000; Abcam), EV71 3D^pol (diluted 1∶10000; self-preparation), and β-actin (diluted 1∶10000; Millipore) antibodies in a WB assay.