Fig 6.

Immunoprecipitation confirms that the MS-detected FLIT peptide in Caski cells originates from 16E5. (A) 16E5 antiserum immunoprecipitates the 16E5 protein. COS cells were transfected with an empty expression vector or with the same vector encoding 16E5 with or without an AU1 epitope tag. Twenty-four hours later, the cells were lysed and immunoprecipitations (IP) were performed (on equal amounts of cell protein) using 16E5 antiserum or preimmune serum from the same rabbit (P.I.). Immunoprecipitated proteins were analyzed on Western blots labeled with an anti-AU1 monoclonal antibody (IB: AU1). (B to F) 16E5 antiserum “clears” Caski cell lysates of FLIT peptide. A Caski cell lysate was digested with trypsin and analyzed by UPLC and MS without immunoprecipitation (B) or following immunoprecipitation with 16E5 antiserum (C) or preimmune serum (E). Proteins immunoprecipitated by the 16E5 antiserum (D) or preimmune serum (F) were eluted, digested with trypsin, and analyzed by UPLC and MS. The black bar indicates the signal intensity of the blank (no sample).