Fig 2: IGFBP-6 production and harvest. a IGFBP-6 expressing U-2OS cells were expanded in DMEM containing 10% fetal bovine serum and subsequently grown in serum-free DMEM. Every 3 days the supernatant was replaced by fresh serum-free medium and again conditioned for 3 more days. The experiment was terminated after 8 cycles of media conditioning, and eight subsequent supernatants (#1 through 8) were collected and analyzed by SDS-PAGE as indicated. Subsequently, the gel was silver stained. M: molecular weight markers. b Supernatants shown in panel a were analyzed by Western blotting, using antibodies to IGFBP-6. Supernatants contain several IGFBP-6 forms of slightly different MW (see below, Fig. 4) which migrated as a broad band in this experiment

Fig 3: IGFBP-6 purification by size exclusion chromatography. a Selected fractions from the gel filtration column were separated by SDS-PAGE along with molecular weight markers (M) and column input (IN), as indicated. The gel was silver stained. b Fractions of the gel filtration column were separated by SDS-PAGE as shown in panel a, transferred to a PVDF membrane, and probed with antibodies to IGFBP-6 using Western blot. Designation of lanes is as in panel a

Fig 4: Functional analysis of purified IGFBP-6. a 450 ng of purified IGFBP-6 and 20 ng of purified IGFBP-3 were separated by SDS-PAGE as indicated, transferred to a PVDF membrane, and probed with biotinylated human IGF-II using an IGFBP Western-ligand blotting kit. Protein species binding to biotinylated IGF-II are indicated in each case. b Diploid human fibroblasts were serum starved for 24 h and then incubated with 50 ng/ml of IGF-II or left untreated, as indicated. Where indicated, 350 ng/ml IGFBP-6 was added. Cellular extracts were prepared and probed with antibodies to Akt, phospho-Akt, and actin, as indicated. The data suggest that IGFBP-6 abrogates IGF receptor dependent signal transduction pathway

Fig 5: IGFBP-6 purification by heparin affinity chromatography. a Selected fractions from the heparin agarose column were separated by SDS-PAGE along with molecular weight markers (M), column input (IN), and column flow-through (FT), as indicated. The gel was silver stained. b Fractions of the heparin column were separated by SDS-PAGE as shown in panel a, transferred to a PVDF membrane, and probed with antibodies to IGFBP-6 using Western blot. Designation of lanes is as in panel a. Please note that elution from the heparin affinity column reveals several distinct forms of IGFBP-6, probably differing in the extent of glycosylation, similar to previous findings with IGFBP-3 (Pircher et al. 2010)