MS2-GFP-Immunoprecipitation (MS2-GFP-IP)

The pcDNA3.1-Axl-minigene plasmid was digested with Xho I, and the linearized plasmid was homologously ligated with the 12×MS2 sequence to construct a pcDNA3.1-Axl-minigene-12×MS2 plasmid. Then, 10 μg of pMS2-GFP and pcDNA-Axl-minigene-MS2-12× plasmid was co-transfected into the corresponding HCCLM3 cells, and the nucleoprotein of liver cancer cells was extracted 48 h later. Subsequently, 3 mg of the cell nucleoprotein was pre-cleared with 50 μl protein G-agarose (Santa Cruz, American) at 4 °C for 2 h. The supernatant was then incubated with GFP antibodies with gentle shaking at 4 °C, overnight. This was followed by the addition of 50 μl of protein A/G-agarose for another 2 h. Lastly, the beads were washed and then re-suspended in 50 μl of 2×SDS-PAGE loading buffer and boiled for 10 min before Western blot detection.