To explore the spectrum of Eya1-interacting partner proteins, we thus generated a stable HEK293 cell line that constitutively express the 2HA–3FLAG-Eya1 (HF-Eya1) protein, purified HF-Eya1 from the cell extracts by a tandem affinity purification protocol to reduce background (first with anti-FLAG and then with anti-HA to purify FLAG-elute) and analyzed 12bands (B1-B12) using mass spectrometry.