Table 3. Pooled data for the binding affinities of GFP–SRY proteins to CaM and Impβ1, as determined using native PAGE and AlphaScreen® assays.
Results in native PAGE experiments such as those in Figure 5(A), were scored according to binding strength: +++, strong binding; +, weak binding; –, no binding. Results are means±S.E.M. for n (in parentheses) experiments for the AlphaScreen® binding experiments (see Figure 5B); where significant differences in binding affinities of the mutant derivatives compared with that of the WT were observed, P values were calculated using Student's t test with InStat2.01. NS, not significant.
| CaM binding | Impβ1 binding | |||
|---|---|---|---|---|
| SRY derivative | Native PAGE | AlphaScreen® Kd (nM) | Native PAGE | AlphaScreen® Kd (nM) |
| WT HMG | +++ (6) | 0.43±0.07 (13) | +++ (6) | 0.94±0.14 (13) |
| HMG-M64T | – (2) | 2.05±0.31 (7) P<0.0001 | +++ (2) | 1.42±0.20 (3) NS |
| HMG-F67V | – (1) | 1.25±0.27 (4) P=0.0004 | +++ (1) | 1.09±0.05 (5) NS |
| HMG-R76P | – (3) | 1.18±0.20 (3) P=0.0004 | +++ (3) | 0.89±0.22 (5) NS |
| HMG-Y127C | +++ (3) | 0.62±0.05 (5) NS | + (3) | 3.60±0.72 (4) P<0.0001 |