Co-immunoprecipitation (Co-IP) assay

A Co-IP assay was performed to determine the relationship between SENP1/HIF1α, HIF1α/SUMO1 and HIF1α/MCT4. The Co-IP kit was used according to the manufacturer's protocol. Briefly, cells seeded in 10-mm dishes were lysed in 1 ml cell lysis buffer. 500 μg proteins were incubated with 2 μg of the indicated antibodies overnight at 4°C under gently rotation, and then they were incubated with 30 μl Protein G-Sepharose beads (Abcam, USA) at 4°C for 2h under rotation. The beads were washed three times with the lysis buffer and resuspended in SDS sample buffer, boiled for 10 min, and then analyzed by immunoblotting procedures described above.