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Fig. 8. (A) Adhesion, not cell spreading, dephosphorylates P-p38. Human keratinocytes null for laminin 5 were suspended and re-adhered to the indicated ligands (laminin 5, collagen) or immobilized mAbs: SP2, an irrelevant non-adhesive mAb; P4C11, a mAb against a 47 kDa non-integrin surface glycoprotein; CD44, a non-integrin cell surface antigen; mAbs against the indicated integrin subunits 
2, 3,6, ß1. Cell adhesion and cell spreading induced by each ligand is indicated. Triton-soluble extracts were prepared from the adherent cells after washing then fractionated by SDS-PAGE and immunoblotted as indicated with antibodies against P-p38, p38 and focal adhesion kinase phosphorylated on Tyr397 (P-FAK). (B) Cytochalasin D inhibits adhesion and dephosphorylation of P-p38 on collagen and fibronectin but not laminin 5. Quiescent laminin 5 null HKs were suspended, treated with cytochalasin D (10 µM), then either left in suspension for 2 hours or incubated on laminin 5, collagen or fibronectin surfaces for 2 hours. Adhesion and spreading of the cells is indicated. Extracts of the indicated cells were blotted with anti-P-p38 and anti-p38 Abs. (C) Re-adhesion of suspended HKs on laminin 5 dephosphorylates P-p38 followed by transient re-phosphorylation. Confluent HKs (with null defects in laminin 5) were suspended using trypsin and either re-adhered to laminin 5 or collagen or suspended over BSA. Triton-soluble extracts were collected from the quiescent adherent HKs, suspended HKs and suspended/re-adherent cells at the indicated intervals (15 minutes, 30 minutes, etc). Expression of P-p38 was examined by immunoblotting. Blots for cells re-adhered on laminin 5 were re-probed with anti-p38 to control for levels of total p38 and are representative of all the blots.
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