Figure 4

(A) Binding of Pb MLSr to ECM by Far-Western blot. PbMLSr (0.5 μg) was subjected to SDS-PAGE and electroblotted. Membranes were reacted with fibronectin (lane 1), type I collagen (lane 2), type IV collagen (lane 3) and laminin (lane 4), and subsequently incubated with rabbit IgG anti-fibronectin, mouse anti-type I and anti-type IV collagen antibodies, and anti-laminin, respectively. Peroxidase-conjugated anti-rabbit and anti-mouse IgG revealed the reactions. Negative control was obtained by incubating PbMLSr with peroxidase-conjugated anti-rabbit IgG (lane 5), and PbMLSr with ECM (lane 6). Positive control was obtained by incubating PbMLSr with polyclonal anti-PbMLSr antibody (lane 7). (B) Binding of PbMLSr to ECM fibronectin, types I and IV collagen (10 μg/mL). The interaction was revealed by ELISA with peroxidase-conjugated streptavidin. The results were expressed in absorbance units. The negative controls were performed using PbMLSr only. (C) Reactivity of PbMLSr to PCM patient sera. 1.0 μg of purified PbMLSr was electrophoresed and reacted to the sera of patients with PCM, diluted 1:100 (lanes 1 to 3) and to control sera, diluted 1:100 (lane 4). The positive control was obtained by incubating PbMLSr with its polyclonal antibody (lane 5). After reaction to the anti-human IgG alkaline phosphatase-coupled antibody (diluted 1:2000), the reaction was developed with BCIP-NBT. (D) Biotinylation assay by Western blot. Lysed A549 cells incubated with biotinylated PbMLSr (lane 1); Lysed A549 cells (lane 2) as negative control.