Affinity purification – mass spectrometry of AFF4 WT or S829/S831/3/5/8A mutant

The S831 phosphosite on AFF4 lies within a disordered region adjacent to the ENL/AF9 binding site. We therefore hypothesized that phosphorylation may regulate protein:protein interactions within the Super Elongation Complex. To investigate this, HEK293T cells expressing RFP negative control, FLAG-tagged AFF4-WT or FLAG-tagged AFF4-A-mutant were stimulated with insulin for 30 min followed by immunoprecipitation and analysis of interacting proteins by LC-MS/MS.