Table 2.
List of clones picked under selective pressure after stable transfection with mutants R15Q, V63I, R142W, 175fs, E186K, E208K, and R220Stop
| Construct | # Clones picked | Surviving clones (%) | Expressors2-a |
|---|---|---|---|
| pREP9 | 41 | 33 (80) | 0 |
| Wild-type | 54 | 41 (76) | 16 |
| R15Q | 68 | 41 (60) | 20 |
| V63I | 72 | 38 (53) | 23 |
| R142W | 80 | 51 (64) | 6 (2 low-moderate)b,c |
| 175fs | 62 | 56 (90) | 0 |
| E186K | 78 | 54 (69) | 9 (4 low-moderate)2-b |
| E208K | 72 | 45 (62) | 26 |
| R220Stop | 52 | 41 (78) | 16 |
Assessment of expression level was based on signal intensity in immunocytochemical analyses and/or on band intensity on immunoblots, relative to that seen with the wild-type clone 517.4.7. As determined through immunocytochemistry, Cx32 expression was heterogeneous in all clones; not all cells in a given population expressed Cx32 protein, even though they were maintained under selective pressure.
Relative to other constructs, few clones expressing these Cx32 mutants were isolated, and the Cx32 expression level in these clones was generally lower and more heterogeneous than the level in clones expressing wild-type or mutant Cx32.
The extent of Cx32 expression in this clone was especially low, accounting for the weak Cx32 bands on immunoblots (see Figure 2).