A, impaired Fyn ubiquitination upon E1 inactivation in Cbl-transfected CHO-TS20 cells. CHO-TS20 cells were transfected with Fyn (0.2 μg) expression plasmid together with 4 μg of GFP or GFP-Cbl plasmids and incubated at 30°C for 56 hr. At this point (To), cells were either maintained at 30°C (30−>30) or shifted to 42°C (30−>42) for the indicated times. Anti-Fyn immunoprecipitates from aliquots of lysate protein (1 mg) were immunoblotted with anti-ubiquitin antibody. B, stabilization of Fyn protein upon E1 inactivation in Cbl-transfected CHO-TS20 cells. Equal amounts (30 μg) of the same cell lysate used in A were immunoblotted with anti-GFP antibody (top panel), anti-Fyn antibody (middle panel), and anti p42/44 MAPK antibody (bottom panel). The levels of Fyn protein were quantified by densitometry, and the values at various times are expressed as a function of the initial Fyn protein level (lane 1) that was assigned a value of 1.0.