Figure 9.

Rescue of Fyn-null oocytes by injection of Fyn cRNA. GV stage oocytes were collected from Fyn-null females following stimulation with PMSG and maintained in GV arrest by culture in the presence of db-cAMP as described in Materials and Methods Section. Oocytes were injected with cRNA encoding c-Fyn or the dominant-negative FynK299M mutant kinase, held in GV arrest for 4–5 hr to allow translation of the exogenous cRNA, then washed free of cAMP and matured for 17 hr before preparation for immunofluorescence. Controls were treated and cultured identically, though not injected with RNA. Samples were stained with FITC-labeled LCA to detect cortical granule contents (left panels labeled green), with alexa 568-phalloidin to stain actin filaments (right panels labeled red), and with to-pro-3 to label DNA (blue). Magnification is indicated by the bar which represents 10 mm. Following immunofluorescence analysis, fixed oocytes were recovered, washed in PBS containing 3 mg/ml PVP, and solubilized in SDS sample buffer containing 20 mM glycine and heated at 90o C for 5 min. Western blot analysis was performed and the blot was probed with anti Fyn-3 antibody (Santa Cruz Biotechnology, Inc.) to detect the presence of Fyn protein in the samples (bottom panel).