Fig. 4. Thrombin cleavage of G4/5 inhibits deposition of pre-lam5 by 3–/– MKs. 3–/– MKs attached to glass coverslips were incubated (overnight) with either culture media alone (A; a-c), media with heparin-purified lam5 (A; d-f) or heparin-purified lam5 that has been digested with thrombin (A; g-i). The coverslips were processed for immunofluorescence to visualize ECM deposits containing the human 2 chain (a,d,g; B4-6), 3 chain (b,e,h; C2-5) or the G4/5 domains of 3²⁰⁰ (c,f,i; D2-1). Deposition occurred only where lam5 containing 3²⁰⁰ (C; lane 1) was added, and was prevented by thrombin digestion of the 3²⁰⁰ to 3¹⁶⁵ (C, lane 2). Bar in (f), 20 µm. (B) Quantitation of deposit area/cell for six random fields for each of the conditions in A. (C) Western blot of lam5 preparations used in A and B. Lane 1, heparin-purified lam5; lane 2, heparin-purified lam5 from lane 1, digested with thrombin.