ADP-Ribosylation Factor 6 Mediates E-Cadherin Recovery by Chemical Chaperones
Figure 4
Arf6 activation or inactivation interferes with E-cadherin expression and localization.
CHO WT, CHO R749W and CHO E757K were transiently transfected with vectors expressing mutant forms of Arf6: ARF6 Q67L (constitutive active form) and ARF6 T27N (dominant negative form). (A) Arf6, E-cadherin and Actin were detected in whole cell lysates by Western Blot. Actin was used as a loading control. The intensity of the bands was quantified and normalized against the transfection control. The intensity average of three independent experiments is shown below the respective sample. (B) Cells were fixed and immunostained with anti-human E-cadherin antibody. Nucleus was counterstained with DAPI. The pictures were taken under a 40× objective. (C) Flow cytometry technique was used to assess E-cadherin cell surface expression. Each histogram represents surface E-cadherin in cells untransfected (yellow), transfected with ARF6 Q67L (blue) or with ARF6 T27N (red) and the transfection control (green). The black area in the histogram represents the cells that were not incubated with primary antibody; this sample was used as negative control. The results are representative of three independent experiments. (D) For each sample, the number of cells expressing surface E-cadherin was calculated. The mean fluorescence intensity was also quantified and normalized against the transfection control of WT expressing cells. The graphs show the average + SE, n = 3 (* represents p≤0.05).