Identification of HEBAlt protein binding partners in thymocytes

In order to identify candidate protein binding partners of the transcription factor HEB, we generated conditional HEBAlt-HA transgenic mice. The transgene consisted of an HA-tagged HEBAlt coding region inserted downstream of a ROSA26-loxP-stop-loxP sequence to enable Cre recombinase activated expression of the transgene. Mice with two transgenic alleles without Cre are termed "TT" whereas those bred onto the Vav-Cre background are termed "TTV". Whole thymocytes were obtained from TT (n=2) or TTV (n=2) mice and subjected to immunoprecipitation using an anti-HA antibody and magnetic beads. Samples were trypsin-digested and analyzed by mass spectrometry on a Q Exactive HF-X Mass Spectrometer by the SPARC BioCentre facility (SickKids, Toronto). Scaffold (version Scaffold_4.8.9, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Protein probabilities were assigned by the Protein Prophet algorithm81.