Fig. 12. Non-cleavable lam5 is deposited while precleaved lam5 is secreted. 3–/– MKs expressing the noncleavable or precleaved human laminin 3 chain were cultured on collagen-coated plates in the presence of ³⁵S-methionine for 3 days. In controls, HFKs were also labeled. Labeled proteins, either secreted into the conditioned culture media (CCM; B) or deposited into the ECM (A) were precipitated with antibody P5H10, against human laminin 3 chain (3), or antibody R5922 against mouse/human laminin ß1 and 1 chains (ß11). The labeled precipitates were fractionated by SDS-PAGE followed by fluorography. (C) Cultures of 3–/– MKs expressing the noncleavable or precleaved human laminin 3 chain were pulse labeled (2 hours) with ³⁵S-methionine. Labeled cells were chased for 0, 3, 8 or 24 hours in media without label. Conditioned culture media (CCM) was immunoprecipitated with antibody P5H10, specific for the human 3 chain, and fractionated by SDS-PAGE (6%, reducing). (D) Cultures of 3–/– MKs or 3–/– MKs expressing noncleavable or precleaved human laminin 3 chain (2.5x10⁶ cells well) were cultured (24 hours) in KGM (1.0 ml). The adherent cells were extracted with Triton X-100 detergent. Total human 3 chain in the ECM and culture medium was quantitated by ELISA with anti-human 3 chain Mab (P5H10). As a control, total laminin ß1 and 1 chains in culture medium were quantitated with anti-ß1/1 chain antibody (R5922). Each analysis was performed on three separate wells.