Fig. 5. Deposition of lam5 into the PBM is inhibited by thrombin removal of G4/5. (A) HFKs were seeded onto confluent cultures of MF on coverslips, cultured for 18-20 hours, then fixed and processed for immunofluorescence with Mabs against the 3 chain (a-c; P5H10) or precursor 3²⁰⁰ (d-f; D2-1). Arrowheads indicate nuclei of HFKs. Thrombin digestion after the culture period removed G4/5 staining in 3²⁰⁰ (e), but 3 was still present in the PBM (b). Thrombin digestion throughout the culture period removed G4/5 from 3²⁰⁰ detected with anti-G4/5 (f) and suppressed deposition lam5 detected with anti-3 (c). Bar in (f), 20 µm. (B) HFKs grown on MFs were double stained with (a) anti-laminin 3 chain (P5H10, green) and (b) anti-integrin ß4 subunit (P4G11, red) and the images overlaid (c, yellow) Bar in (c), 20 µm. (C) ELISA quantitation of lam5 deposits. After the designated time periods (5, 10, 20 hours), the HFK/MF co-cultures were processed for ELISA using anti-2 Mab (B4-6). The amount of lam5 detected was significantly lower for all time points when thrombin was included during the co-culture (thrombin). Hirudin, a thrombin inhibitor, prevented the effects of thrombin.