Immunoprecipitation of NAP1-GFP

Transiently transfect FIP200 knockout HAP1 cells with pcDNA3.1 NAP1-EGFP (Addgene), or empty pcDNA3.1 vector as a negative control, using Lipofectamine 2000 (Thermo Fisher).

After 48 h, collect the cells by trypsinization and wash the cell pellet with PBS once. Then, lyse the cells in lysis buffer.

Lyse the samples for 0h 20m 0s On ice. Then, clear the cell lysates by centrifugation at 20000x g,4°C.

Determine the protein concentrations of the cleared protein lysates with the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher).

For both samples, negative control and NAP1-EGFP lysates, incubate 12mg of cell lysate 0h 20m 0s with 20µL or GFP-Trap agarose beads (GTA-20, Chromotek).

In the morning, wash the samples three times in lysis buffer. Then, resuspend the beads in protein loading dye, supplemented with 100 mM DTT. Finally, boil the samples for 0h 5m 0s at 95°C.

Load the samples on 4-12% SDS-PAGE gels (NP0322BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher).

After the run, stain the SDS-PAGE gel with Coomassie, fix for 0h 10m 0s in fixation solution, and destain it 0h 5m 0s in dH2O.

Cut the band corresponding to NAP1-EGFP from the gel with a fresh scalpel and submit for mass spectrometry analysis.