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Fig. 10. Deposits of recombinant lam5 direct assembly of integrin ß4 in detergent-resistant SACs. (A) 3+/+ MKs (a-b), or 3–/– MKs expressing noncleavable human laminin 3 chain (e-f and i-j), or precleaved 3 chain (g-h and k-l). Cells were cultured on BSA-coated glass coverslips for 24 hours. 3–/– MKs (c-d), which cannot attach to BSA-coated surfaces, were cultured on collagen-coated coverslips. Cultures were extracted with 0.25% Triton X-100 in PBS, fixed and examined by immunofluorescence microscopy. Double staining with anti-integrin ß4 (Mab P4G11; panels b, d, f, h, j and l) and Mabs against: mouse/human laminin 3 (D3-4; a and c), human laminin 3 chain (P5H10; e and g), or against G4/5 in human laminin 3-200 (D2-1;i and k). a-b, c-d, e-f, g-h, i-j, and k-l are each the same field. Bar, 10 µm. (B) Quantitation by ELISA of human laminin 3 chain deposition (Mab P5H10) and mouse laminin 3 chain (Mab p3C8), and Triton X-100 resistant mouse integrin ß4 in Triton extracted cultures of 3–/– MKs, 3+/+ MKs or 3–/– MKs expressing noncleavable (non-cl.) or precleaved (pre-cl.) human laminin 3 chain.

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