Fig. 3. Co-immunoprecipitation of ECR3E and TGF-ß activity from primary keratinocytes. Wild-type and transgenic (ECR3E) primary keratinocytes were isolated and grown in low calcium medium. Conditioned medium was collected and used in immunoprecipitation (IP) experiments with either anti-Myc (A14) or anti-LTBP (Ab39) antibodies. Nonimmune rabbit-IgG (RIgG) or rabbit serum (RS) were used as negative controls. Immunoprecipitates were analyzed for TGF-ß activity using the PAI-1-luciferase assay as described in Materials and Methods. PAI-1-luciferase transfected mink lung cells were grown for 16 hours in the presence of heat-activated wild-type (wt) or transgenic (tg) immunoprecipitates in the absence or in the presence of LAP or neutralizing isoform-specific anti-TGF-ß antibodies (anti-TGF-ß1, anti-TGF-ß2 and anti-TGF-ß3).