Table 2. Experimental conditions used in steady state and actomyosin contraction assays.
50 mM HEPES pH 7.3, 1 mM MgCl2, 1 mM TCEP, 1 mg / ml BSA, and 0.2 mg / ml glucose oxidase +0.035 mg / ml catalase +1 mM glucose (O2 scavenging system) were included in every assay.
| Experiment | His8-pLAT | His10- Ezrin (ABD) |
Grb2 | Sos1 | pSLP-76 | Nck | N-wasp | WASP | Actin | Myosin II | KCl | ATP |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| pLAT → Sos1 (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125nM | 125nM | 0nM | 0nM | 0nM | 0nM | 0nM | 0nM | 75mM | 1mM |
| pLAT → Sos1 +Actin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
125 nM |
0 nM |
0 nM |
0 nM |
0 nM |
250 nM |
0 nM |
75 mM |
1 mM |
| pLAT → Sos1 +Actin + Myosin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
125 nM |
0 nM |
0 nM |
0 nM |
0 nM |
250 nM |
100 nM |
75 mM |
1 mM |
| pLAT → pSLP-76 (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
0 nM |
0 nM |
0 nM |
0 nM |
0 nM |
75 mM |
1 mM |
| pLAT → pSLP-76 +Actin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
0 nM |
0 nM |
0 nM |
250 nM |
0 nM |
75 mM |
1 mM |
| pLAT → pSLP-76 +Actin + Myosin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
0 nM |
0 nM |
0 nM |
250 nM |
100 nM |
75 mM |
1 mM |
| pLAT → Nck (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
0 nM |
0 nM |
0 nM |
0 nM |
75 mM |
1 mM |
| pLAT → Nck +Actin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
0 nM |
0 nM |
250 nM |
0 nM |
75 mM |
1 mM |
| pLAT → Nck +Actin + Myosin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
0 nM |
0 nM |
250 nM |
100 nM |
75 mM |
1 mM |
| pLAT → N-WASP (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
125 nM |
0 nM |
0 nM |
0 nM |
75 mM |
1 mM |
| pLAT → N-WASP +Actin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
125 nM |
0 nM |
250 nM |
0 nM |
75 mM |
1 mM |
| pLAT → N-WASP +Actin + Myosin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
125 nM |
0 nM |
250 nM |
100 nM |
75 mM |
1 mM |
| pLAT → Sos1 +Actin + Myosin (Contraction) For Grb2, Grb2Basic |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
125 nM |
0 nM |
0 nM |
0 nM |
0 nM |
250 nM |
100 nM |
50 mM |
0.05 mM |
| pLAT → N-WASP +Actin + Myosin (Contraction) For N-WASPWT, N-WASPNeutral, N-WASPBasic |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
125 nM |
0 nM |
250 nM |
100 nM |
50 mM |
0.05 mM |
| pLAT → WASP (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
0 nM |
125 nM |
0 nM |
0 nM |
75 mM |
1 mM |
| pLAT → WASP +Actin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
0 nM |
125 nM |
250 nM |
0 nM |
75 mM |
1 mM |
| pLAT → WASP +Actin + Myosin (Steady-State) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
0 nM |
125 nM |
250 nM |
100 nM |
75 mM |
1 mM |
| pLAT → WASP +Actin + Myosin (Contraction) |
20 nM (sol’n) 500 molecules /μm2 |
five nM (sol’n) ~100 molecules /μm2 |
125 nM |
62.5 nM |
62.5 nM |
125 nM |
0 nM |
125 nM |
250 nM |
100 nM |
50 mM |
0.05 mM |