TABLE 1.
Effect of ASAP1 mutants on podosome formation
| Recombinant protein | % of cells with podosomes (no. of expts)a
|
|
|---|---|---|
| −siRNA | +siRNA | |
| None | 38 ± 5 (7) | 6 ± 4 (6)** |
| ASAP1a | 35 ± 4 (7) | 31 ± 3 (6) |
| ASAP1b | 46 ± 10 (5) | 38 ± 7 (4) |
| ASAP1bb | 30 ± 3 (5) | 30 ± 2 (4) |
| [R497K]ASAP1b | 42 ± 10 (5) | 33 ± 5 (4) |
| [Y782F]ASAP1b | 2 ± 3 (5)** | 4 ± 5 (4)** |
| [R811A]ASAP1b | 29 ± 3 (5) | 32 ± 5 (3) |
| [R1021A]ASAP1b | 38 ± 7 (5) | 29 ± 8 (4) |
| [Y1094A]ASAP1a | 24 ± 4 (7)** | 22 ± 3 (6)* |
| [E1103A]ASAP1a | 23 ± 7 (7)** | 22 ± 10 (6)* |
| [W1122A]ASAP1a | 13 ± 1 (2)** | 12 ± 1 (2)** |
| [ΔBAR]ASAP1b | 31 ± 4 (5) | 15 ± 6 (4)** |
| [ΔSH3]ASAP1b | 4 ± 3 (5)** | 7 ± 3 (4)** |
| [817-873]ASAP1a | 25 ± 5 (3)* | ND |
Podosomes were visualized in NIH 3T3 fibroblasts expressing [Y527F]c-Src and the indicated recombinant ASAP1 protein. Where indicated, the fibroblasts were left untreated (−siRNA) or were treated with siRNA (+siRNA) targeting endogenous ASAP1 but not the RNA for the recombinant proteins. Cells were scored for the presence of podosomes. At least 30 cells were counted for each experiment. The values presented are means ± standard deviations. The data were analyzed by analysis of variance, followed by Dunnett's multiple-comparison test. For the first column, significant differences from the value for no recombinant ASAP1 are indicated by asterisks; for the second column, significant differences from the value for cells expressing ASAP1a are indicated by asterisks (*, P < 0.05; **, P < 0.01). ND, not determined.
Expressed using the pSI plasmid resulting in levels less than those achieved with the pCI plasmid used for all other constructs presented in this table (Fig. 5B).