Co-immunoprecipitation of ERK1/2 and STAT3

The total protein was extracted as mentioned before. Co-IP was conducted as described previously [27 (link)]. 10% of the supernatants were prepared for Input, and the rest were incubated with normal IgG, anti-ERK1/2 (dilution 1:20), or anti-STAT3 (dilution 1:20) at 4 °C overnight with gentle end-over-end mixing, followed by pre-washed Protein A/G magnetic beads (Shanghai Epizyme Biomedical Technology Co., Ltd), mellow agitation, and overnight attachment. Protein A/G magnetic beads conjugates were separated by a magnetic rack. Proteins were eluted by boiling with loading buffer and then detached from beads by the magnetic rack.