Fig. 5.
Activated ARF6 partially colocalizes with activated ERK in invadopodia, and the ARF6 GTPase cycle regulates ERK activation. Cells were transfected as indicated and seeded on CFDSE-labeled gelatin and allowed to invade. Cells were immunofluorescently labeled for HA and/or phosphorylated ERK as indicated. These images were pseudocolored for better visualization of colocalization between ARF6 (red) and phospho-ERK (green). CFDSE-labeled gelatin is pseudocolored blue. (A and B) Images shown here are single confocal sections at the tips of invadopodia of invading ARF6(Q67L)-expressing cells. A is the same as B except that A does not show gelatin staining. (C–H) These images are confocal sections taken along the cell body to better visualize the differences in phospho-ERK levels. Cells were transfected with ARF6(Q67L)-HA (C–F) or ARF6(T27N)-HA (D–G) and labeled for HA or phospho-ERK as indicated. As seen, ARF6(Q67L)-transfected cells have increased levels of activated ERK (arrows), whereas ARF6(T27N)-transfected cells have decreased levels of activated ERK (arrows) compared with other untransfected cells in the same field. (Bar = 10 μm.) (I) LOX cells transfected with plasmids expressing ARF6(Q67L) or ARF6(T27N) or transfected with empty plasmid as control, were plated on gelatin-coated tissue culture dishes, and treated with or without 40 ng/ml HGF for 30 min and lysed. Equal amounts of cell lysates were resolved by SDS/PAGE followed by probing with antisera directed specifically against total ERK, phospho-ERK, or HA. Representative immunoblots of three independent experiments are shown. The relative intensity of lower phospho-ERK band (arrow) was assessed by densitometric scanning.