Fig. 1.

Immunoprecipitation of tristetraprolin (TTP) and identification of poly(A) binding protein (PABP) fragments by mass spectrometry. Epitope-tagged TTP was expressed in transfected BHK and NIH3T3 cells and immunoprecipitated for phosphorylation site analysis (as described in Ref. 10). Peptides from a number of proteins involved in mRNA metabolism were reproducibly recovered in significant quantity only in the TTP-expressed samples (A). A significant percentage of the sequenced peptides were found to be PABP, surprising since the role of TTP is to promote deadenylation and therefore destroy the PABP binding site. Polyadenylate-binding protein 1 (PABP1) protein sequence and peptide coverage (bold and underlined; B) routinely detected from analyses of tryptic digests by online nHPLC-μESI mass spectrometry. Many of these are shared by more than 1 PABP isoform. Peptides that are unique to PABP1 and PABP4 are shown in C and D, respectively.