Fig. 7. Presence of phosphate groups on Ser-38 and Ser-72 affect filament forming ability following microinjection into BHK-21 fibroblasts. (A) Wild-type (a-c), PKA-phosphorylated wild-type supernatant fraction (d-f), and S38:A/S72:A (g-i) myc-tagged vimentin were microinjected at a concentration of 1 mg/ml into BHK-21 fibroblasts. At 10 minutes (a, d, g), 30 minutes (b, e, h) and 3 hours (c, f, i) post-injection, cells were fixed with methanol and indirect immunofluorescence was performed using a monoclonal antibody directed against the myc-tag to trace the microinjected protein. Filament formation by the injected protein was assessed, and confocal micrographs were obtained, demonstrating the degree of filament formation observed for the majority of cells at a given time point. (B) Co-localization of the injected protein with the endogenous BHK-21 IF network was determined at 3 hours post-injection by double-label immunofluorescence using a polyclonal antibody specific for the endogenous vimentin (a, b, c). The images show the behavior of the S38:A/S72:A mutant pelletable protein but similar results were obtained with all three injected proteins.