Metabolic labeling of IGFBP-5 by T47D breast carcinoma cells Metabolic labeling of IGFBP-5 by T47D breast carcinoma cells.a, cells were cultured in the presence of [32P]orthophosphoric acid, and IGFBP-5 in conditioned media was detected by Western immunoblotting (WIB) and autoradiography after SDS-PAGE. b, conditioned media from non-radioactive replicate wells were collected at time points up to 12 h, and levels of secreted IGFBP-2 (open bars) and -5 (closed bars) were quantitated by RIA. c, T47D breast carcinoma cell-derived IGFBPs were purified by IGF-I affinity chromatography and then fractionated by reverse-phase HPLC. UV absorbance is indicated on the left axis, and percent Phase B (60% acetonitrile in 0.1% TFA) is indicated on the right axis. Metabolic labeling of IGFBP-5 by T47D breast carcinoma cells.a, cells were cultured in the presence of [32P]orthophosphoric acid, and IGFBP-5 in conditioned media was detected by Western immunoblotting (WIB) and autoradiography after SDS-PAGE. b, conditioned media from non-radioactive replicate wells were collected at time points up to 12 h, and levels of secreted IGFBP-2 (open bars) and -5 (closed bars) were quantitated by RIA. c, T47D breast carcinoma cell-derived IGFBPs were purified by IGF-I affinity chromatography and then fractionated by reverse-phase HPLC. UV absorbance is indicated on the left axis, and percent Phase B (60% acetonitrile in 0.1% TFA) is indicated on the right axis. An autoradiograph of a ligand blot of the HPLC fractions 26–37 inclusive using 125I-IGF-I is shown in the inset and overlaid at the appropriate retention times. Mark E. Graham et al. Mol Cell Proteomics 2007;6:1392-1405 © 2007 The American Society for Biochemistry and Molecular Biology