Table 1.
Relative protein expression and effect of inhibitors on cell lines and recombinant Arf GEFs and Arf GAPs used in this study
| Cell line or recombinant protein | Arf6 expression (relative to U2OS) | Brag2 expression (relative to NIH3T3) | ASAP1 expression (relative to NIH3T3) | EC50 for cell killing (μM) |
||||
|---|---|---|---|---|---|---|---|---|
| NAV-2729 | Brefeldin A | Bragsin | Bragsin2 | SecinH3 | ||||
| OSA | 0.17 | 0.20 | 0.03 | 9 ± 2 (n = 3) | 0.026 ± 0.004 | >50 | >50 | >50 |
| U2OS | 1.00 | 0.51 | 0.37 | 9 ± 1 (n = 3) | 0.024 ± 0.002 | >50 | >50 | >50 |
| RD | 0.11 | 0.44 | 0.20 | 8 ± 4 (n = 3) | 0.006 ± 0.002 | >50 | >50 | |
| SMS-CTR | 0.71 | 0.77 | 0.14 | 11 ± 5 (n = 3) | 0.016 ± 0.002 | >50 | >50 | |
| HeLa | 0.30 | 0.49 | 0.02 | 9 ± 2 (n = 3) | 0.04 ± 0.01 | >50 | >50 | |
| MCF 10A | 0.58 | 0.89 | 0.05 | 10 ± 4 (n = 3) | 0.11 ± 0.04 | >50 | >50 | |
| NIH3T3 | 0.71 | 1.00 | 1.00 | 11 ± 3 (n = 3) | 0.44 ± 0.14$$ | >50 | >50 | |
| C2C12 | 0.43 | 0.43 | 0.95 | 11 ± 2 (n = 3) | 0.46 ± 0.07$$ | >50 | >50 | |
| IC50 for protein inhibition (μM) | ||||||||
| Brag2Sec7-PH with LUV | 7.1 ± 2.5 (n = 3) | 31 ± 12 (n = 3)∗ | 25 ± 10 (n = 4) | |||||
| ARNOSec7-PH with LUV | 37 ± 11 (n = 4)∗∗ | |||||||
| AGAP1 with LUV | 2.7 ± 1.4 (n = 5) | |||||||
| ARAP1PPZA with LUV | >50 (n = 3) | |||||||
| ArfGAP1 with LUV | >50 (n = 3) | |||||||
| ASAP1PZA with LUV | 4.6 ± 0.9 (n = 4) | |||||||
| ASAP3PZA with LUV | 9.1 ± 3.2 (n = 4)#,&& | |||||||
| ASAP1PZA with diC8-PIP2, L8K | 29 ± 6.7 (n = 6)#### | |||||||
| ASAP1 with LUV | 12.2 ± 1.4 (n = 4) | |||||||
| ASAP1PZA with LUV (2.5% PIP2), L8K | 13.4 ± 2.3 (n = 4) | |||||||
Top. $$, different than human cells, p < 0.01. ANOVA with Tukey post hoc multiple comparison test. Proteins were detected by immunoblotting. Signal was normalized to GAPDH or vinculin and is expressed as a fraction of the maximum signal observed. Twenty micrograms protein of the total cell lysate were loaded on the gels. Bottom. IC50s for recombinant protein are means ± SDs. ∗, different than NAV-2729 for inhibition of Brag2, p < 0.05; ∗∗, different than inhibition of Brag2, p < 0.01 by ANOVA analysis of Brag2 and ARNO data with Tukey post hoc multiple comparison test. #, greater than ASAP1 with LUV, p < 0.05, ####, greater than ASAP1 with LUV, p < 0.0001, &&, greater than AGAP1 with LUV, p < 0.01, determined by one way ANOVA followed by a Tukey multi comparison test for AGAP1 with LUV, ASAP1 with LUV, ASAP1 with diC8-PIP2, and ASAP3 with LUV.