Figure 4. Tris DBA inhibits ARF6 activity and localizes B-catenin to plasma membrane.
(A) ARF6-GTP pulldown assay was performed on in vitro Mel92.1 and Mel202 cell lines to collect protein indicative of AFR6 activity. Protein collected from pulldown assay was then used to perform Western Blot analysis and compared to DMSO control protein levels which revealed significant inhibition of ARF6 activity at 3 uM in the Mel92.1 cell line and 5 uM in the Mel202 cell line. (B) Plasma membrane and total membrane fractions isolated from 92.1cells treated with no drug or Tris DBA for 24 hours at 2 μM concentration. Cells were fractionated to collect plasma membrane. Western Blot of B-catenin and GNAQ is shown. Na,K-ATPase was used as loading control for plasma membrane fractions and GAPDH was used as the loading control for cytosol fractions. Briefly, 100 × 106 cells treated with no drug or Tris DBA were collected and homogenized. Cells were centrifuged and the supernatant (cytosol) was collected. The remaining pellet was purified according to manufacturer instructions (Abcam) for plasma membrane fractions. Quantitation of B-catenin and GNAQ from plasma fractions was performed using Image Studio Lite from LI-COR Biosciences.