SDS-PAGE of IGFBP-5 from 32P-labeled T47D cells and separation of tryptic phosphopeptides separated by HPLC.a, an autoradiograph (lane 1) is shown next.

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binding sites 58 of the 473 unambiguously assigned phosphorylation sites are predicted by Scansite to be sites for binding. 50 of these correspond.

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SDS-PAGE of IGFBP-5 from 32P-labeled T47D cells and separation of tryptic phosphopeptides separated by HPLC.a, an autoradiograph (lane 1) is shown next to 3 μg of the Coomassie-stained protein (lane 2). b, a total of 15 μg of IGFBP-5 was digested with trypsin and applied to a reverse-phase column. SDS-PAGE of IGFBP-5 from 32P-labeled T47D cells and separation of tryptic phosphopeptides separated by HPLC.a, an autoradiograph (lane 1) is shown next to 3 μg of the Coomassie-stained protein (lane 2). b, a total of 15 μg of IGFBP-5 was digested with trypsin and applied to a reverse-phase column. Fractions were collected, and the radiation was measured in each fraction as shown on the left axis. The percent increase in concentration of organic phase (Phase B) is shown on the right axis. Each phosphopeptide detected by MALDI-TOF MS and dephosphorylated in vitro by phosphatase (Table I and “Experimental Procedures”) was matched to the IGFBP-5 sequence by molecular mass. The matches to amino acids in the IGFBP-5 sequence are shown for each fraction in which they were detected. An IGFBP-2 phosphopeptide was also detected in fractions 29 and 30 as shown. c, the sequence of mature human IGFBP-5 from Swiss-Prot accession number P24593 without the signal sequence, i.e. the first 20 amino acids. The in vivo phosphorylation sites at Ser96 and Ser248, determined in this work, are shown in white on black. The known glycosylation site at Thr152 is underlined. Leu1, which was also shown in this work to be absent in a portion of the mature chain from T47D cells, is shaded. Putative heparin-binding motifs are indicated in boxes. Mark E. Graham et al. Mol Cell Proteomics 2007;6:1392-1405 © 2007 The American Society for Biochemistry and Molecular Biology