Table III.
Quantitative Analysis of Coimmunoprecipitation of Fyn with CD8-ζ
| Construct | Relative coimmunoprecipitation | ||||
|---|---|---|---|---|---|
| CD8-ζ | + Fyn | 100% | |||
| + K299M-Fyn | 35% | ||||
| + ΔSH2-Fyn | 14% | ||||
| + R176K-Fyn | 38% | ||||
| CD8-ζT76 | + Fyn | 91% | |||
| CD8-ζ4F | + Fyn | 19% | |||
| CD8-ζ + Fyn | (Octylglucoside) | 14% | |||
| (Filipin) | 54% | ||||
| CD8-ζ | + FynKRas | 93% | |||
| + G2A,C3S-FynKRas | 26% | ||||
COS-1 cells were transfected with cDNAs encoding the indicated constructs, and coimmunoprecipitation experiments were performed as described in Fig. 5. In each experiment, coimmunoprecipitation of Fyn protein constract with anti–CD8 antibody was measured in duplicate and multiple films representing different exposure times to the blot were used to ensure linarity of the signal. These values were first corrected for the total level of Fyn protein in each immunoprecipitation, as measured by the second Fyn immunoprecipitation step (see Materials and Methods). Given numbers for each test are expressed relative to the amount of wild-type Fyn that coimmunoprecipitates with full length CD8-ζ and represent the mean of two experiments, with errors ranging from 5 to 15%. The given 100% value of wild-type Fyn coimmunoprecipitating with full length CD8-ζ represents 10–15% of total Fyn expressed (see Fig. 7).