Fig. 5. Effect of PAPPA on IGF-mediated growth responses in mouse embryonic fibroblasts. (A) Fibroblasts from wild-type (WT) and PAPPA^-/- embryos were incubated for 48 hours prior to the addition of recombinant wild-type IGFBP4 (lanes 3, 4) or protease-resistant IGFBP4 (lanes 5, 6). Lanes 1, 2 are cells with no IGFBP4 added. After an additional 1 hour of incubation, cells were lysed and immunoprecipitated with either antibody against the type I IGF receptor (even number lanes) or non-specific IgG (odd number lanes), as described in the Materials and methods. Immunoprecipitates were run on SDS-PAGE under reducing conditions and immunoblotted with phosphotyrosine antibody (Tyr-P IGF1R) or type 2GF receptor antibody (IGF1R). (B) Fibroblasts from WT (n=5) and PAPPA^-/- embryos (n=8) were incubated for 48 hours prior to the addition of 25 nM IGFBP4 and 5 nM IGF (black bars) or IGF analog (gray bars). Results of experiments using IGF1 and IGF2 [and corresponding analogs, des(1-3)IGF1 and des(1-6)IGF2] were combined as they gave equivalent responses (see Table 2). Addition of 25 nM IGFBP3 and 5 nM IGF is represented by the white bars. [³H]Thymidine incorporation was measured as described in the Materials and methods. Data (mean±s.e.m.) are expressed as percent of control. ^*, significantly different from control, P<0.01.