Fig. 4. hLTBP-3 protein is expressed in transfected COS-7 cells, and its secretion is affected by co-expression of TGF-ß1. (A) COS-7 cells were plated on glass coverslips and transfected with the full-length expression construct of hLTBP-3. The cells were fixed three days after transfection and subjected to immunofluorescence analysis using the affinity-purified hLTBP-3 antibody (Ab-hL3/2925). White arrows and outlining are used to mark the two cells showing an intense fluorescent signal of the translation product of hLTBP-3. (B) COS-7 cells were transfected with the cDNA expression constructs indicated on the figure. Conditioned cell culture medium was collected 3 days after transfection and analyzed by immunoblotting. The proteins were separated by 7.5% SDS-PAGE under non-reducing conditions and subjected to immunoblot analysis using an anti-hLTBP-3 (Ab-hL3/2925) antibody. An arrow indicates the 240 kDa complexes, which are secreted when either of the indicated two full-length hLTBP-3 cDNAs and pTGF-ß1 are co-transfected. An arrowhead on the left indicates the smaller complex, which is secreted following the co-transfection of phL3/699-1153 expression construct and pTGF-ß1. The same symbols are used in C. (C) Similar complexes recognized by an antibody specific to TGF-ß1•LAP. Immunoblotting was carried out as defined above with the exception that anti-ß1•LAP antibody (680) was used in immunodetection. Free ß1•LAP and unprocessed TGF-ß1•LAP dimers are marked by a bracket. (D) Immunoprecipitation analysis of the conditioned medium of transiently transfected COS-7 cells using anti-hLTBP-3 (Ab-hL3/2925) antibody. The samples were separated by SDS-PAGE using 4-15% gradient gels under reducing conditions.