Fig. 8. Paxillin overexpression inhibits NMuMG cell motility. NMuMG cells were retrovirally transduced with GFP, GFP-FL pax and GFP pax . (A) Indirect immunofluorescence microscopy (a-f) of NMuMG cells expressing either GFP, GFP-FL pax or GFP-pax . Arrows (b,d,f) indicate phospho-paxillin Y118 staining in focal adhesions along the periphery of epithelial islands is suppressed in GFP-pax expressing cells. (B) Detergent-soluble lysates of retrovirally transduced NMuMG cells untreated (–) or treated with TGF-ß1 (+) for 48 hours expressing either GFP, GFP-FL pax or GFP-pax were prepared and run on standard SDS-PAGE. Western blot analysis was performed utilizing antibodies to the specified proteins. (C) NMuMG cells retrovirally transduced and treated with TGF-ß1 for 48 hours followed by GFP immunoprecipitation. Immunoblot analysis was performed using antibodies to GFP, phosphotyrosine (clone 4G10) or phospho-paxillin Y118. Arrowhead indicates GFP-FL pax and arrow indicates GFP-pax . (D) Western blot and modified Boyden chamber migration assays were performed with retrovirally transduced NMuMG cells expressing GFP, GFP-FL pax, GFP-pax or GFP-Hic-5. Immunoblots were performed with antibodies to GFP and FAK. Cells were allowed to migrate to 10 µg/ml fibronectin for 16 hours followed by fixation, staining and quantification of cells on the underside of the filter. Experiments were performed in triplicate. Migration is represented as a percentage of the GFP control with significance levels *P<0.05 and **P<0.001. Bar, 5 µm.