Fig. 7. The role of matrix molecules and their associated proteins in stretching-mediated signaling. AECs maintained for 4 days on elastomer membranes were subjected to 10% stretching at 30 cycles per minute for 10 minutes. (A) Immunoblots of extracts of AECs, either unstretched (–) or subjected to the stretch regimen (+), probed first with antibodies against phosphorylated MAPK. The same blots were then reprobed with antibodies against total MAPK. The blots are representative of at least three separate trials. The static and stretched cells were incubated in the presence (+) or absence (–) of antibodies that functionally inhibit the 3 laminin subunit (50 µg ml^–1), 3 integrin (50 µg ml^–1), ß1 integrin (50 µg ml^–1) or dystroglycan (40 µg ml^–1), as indicated, for 18 hours before stretching. The bar graphs in B are quantifications of the densitometric scans of immunoblots of extracts derived from AECs in three separate trials following 10% stretching at 30 cycles per minute for 10 minutes in the presence of various cell-surface and matrix antibody antagonists or reagents, or following infection with adenovirus encoding either control or dystroglycan shRNA. Blots were probed first with antibodies against activated p42/p44 MAPK and then with antibodies against total p42/p44 MAPK. In each case, the extent of phosphorylation of p42/p44 MAPK in stretched AECs was normalized to the total level of MAPK in the same sample. The percentage phosphorylation was calculated relative to untreated, stretched values in each set of studies. Error bars indicate standard deviations. (C) AECs were infected with adenovirus encoding either control or dystroglycan shRNA at 2 days after plating. 24 hours later, the cells were washed and incubated for 18 hours in serum-free medium. Extracts of uninfected and infected cells were then prepared for SDS-PAGE and immunoblotting using a monoclonal antibody against ß-dystroglycan. The same immunoblot was then reprobed with an antibody against actin.