Western Blot Analysis and Co-immunoprecipitation (Co-IP) Assay

Proteins were extracted using RIPA buffer (Solarbio). The concentration of proteins of the supernatant was determined using a BCA assay kit (Thermo Scientific). The proteins (90 μg per well) were separated and transferred into PVDF membranes (Millipore). Then, the membranes were incubated with rabbit polyclonal antibody to DDX5 (1:1000, Abcam), rabbit ployclonal antibody to Cyclin E1 (1:800, Anbo), rabbit polyclonal antibody to TCF12 (1:600, Millipore), and mouse monoclonal antibody to GAPDH (1:2000, Proteintech Group), according to protocols in our previously published articles (Liu et al., 2014; Wang et al., 2017).

The Co-IP assay was performed by Pure Proteome^TM Protein A/G Mix Magnetic Beads (Merck) following the manufacturer’s instructions. Briefly, part of the extracted and quantified total proteins was harvested as the positive control and labeled as “input.” Equal amounts of protein, 500 μg proteins in 400 μL supernatants, were incubated with 4 μg DDX5 antibody (Abcam) or TCF12 antibody (Millipore) and Protein A/G with overnight rotation at 4°C. The immune complexes were harvested and labeled as “Co-IP.” Then, all the “Co-IP” and “input” proteins were analyzed by Western blot analysis using the indicated primary antibodies.