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Fig. 6. 3ß1 is required for adhesion-dependent activation of ERK. (A) Wild-type MK^+/+ cells (+/+ lanes), 3-null MK^-/- cells (-/- lanes), or 3-transfected MK^-/- cells (3 lanes) were kept in suspension for 30 minutes or adhered to LN-5 ECM under serum-free conditions for 15 minutes or 24 hours, as indicated. To assay ERK phosphorylation, cell lysates were immunoblotted with antibodies against phosphorylated ERK (pERK, upper panel) or total ERK (ERK, lower panel). (B) To assay ERK activity, lysates were prepared from suspended cells or from cells adhered to LN-5 for 15 minutes, then tested for in vitro phosphorylation of an Elk-1 substrate (pELK), as described in the methods. (C) MK^+/+ cells were adhered to LN-5 ECM and cultured in serum-free medium for 24 hours in the absence of antibody (no Ab), or in the presence of either anti-6 blocking antibody (GoH3) or rat IgG_2a isotype control antibody (IgG), as described in Fig. 3. Cell lysates were immunoblotted with antibodies against phosphorylated ERK or total ERK, as described in A.

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