Figure 4

Laminin γ2 short arm drives deposition of laminin 5 to the ECM. Immunofluorescence analysis of control (A) and split skin (B) using pAb SE1097. The strong linear staining indicates that the 440-kD form of laminin 5 and/or the NH₂-terminal cleaved γ2 polypeptide are present in human basement membrane. Medium: 50 μg of ECM (lane 1), proteins of spent medium (lane 2), and total cellular extracts (lane 3) obtained from cultures of normal HNKs were analyzed by immunoblotting using pAb SE144 directed against the COOH-terminal domains of the laminin γ2 chain (C), and pAb SE1097, specific to the NH₂-terminal domains IV and V of the γ2 short arm (D). The mass of molecular markers is indicated on the left of the gels. (C) In the ECM the migration positions of the laminin γ2 polypeptides show the major presence of the cleaved form of the laminin 5 γ2 chain in comparison to the noncleaved form. (D) Reaction with pAb SE1097 reveals the presence of the 50-kD product of proteolytic cleavage of the γ2 chain in the matrix and in the spent medium. (E) Immunofluorescence analysis of HKSV keratinocytes expressing the recombinant short arms of the laminin chains γ2 (pγ50; a and b) and β3 (pβ60; c and d). Double immunofluorescence labeling performed using either mAb T7 (a) or mAb anti-HA (c) and phalloidin (b and d) demonstrates that the recombinant γ2 short arm is deposited into the ECM (E), whereas the recombinant β3 short arm is not detected in the ECM (G). Bars: (A) 50 μm; (E, d) 20 μm.