Fig. 5.

Proteomic dissection of Tec tyrosine kinase signaling. A: Tec-associated proteins were purified by FLAG tag-based chromatography (left) or immunoprecipitation (IP; right). Cells transfected with nontagged Tec (NT) were used as a negative control. Proteins were separated by SDS-PAGE, and gels were stained with SYPRO. B: workflow for proteomic analyses. Human embryonic kidney-293 cells were transfected with pcDNA3 containing the Tec-COOH-FLAG insert (or pcDNA3 with untagged Tec as a control). Forty-eight hours later, cells were lysed and subjected to either FLAG IP using anti-FLAG-M2 antibody or column purification with FLAG affinity beads. Tec-associated proteins were separated by SDS-PAGE and stained with Coomassie blue. Protein bands were excised, digested with trypsin, and analyzed with liquid chromotography (LC)/mass spectrometry (MS)/MS on an Orbi-trap. Protein identification was carried out by database searching using the Sequest algorithm. Results from both purification processes were merged and then filtered.