Overexpression of Git2-short, but not its
GAP-inactive mutant, causes a redistribution of β-COP. HeLa cells
were transiently transfected with 4 μg of pcDNA3 plasmids encoding
HA-ARF1N126I (a and b), ARF6N122I (c and d), HA-Git2-short (e and f),
the CA mutant (g and h), or 2 μg each of HA-Git2-short and
myc-ARF1Q71L (i and j), using the calcium phosphate method. After
48 h, cells were fixed and HA-ARFs or HA-Git2-short proteins were
visualized using mouse anti-HA antibody and Cy3 anti-mouse IgG (a, c,
e, and g). Endogenous β-COP was visualized with rabbit anti-β-COP
antibody and Cy2 anti-rabbit IgG (b, d, f, h, and j). In i,
HA-Git2-short was visualized using biotin-labeled mouse
monoclonal anti-HA antibody and Cy3 streptavidin (but shown in green),
and myc-ARF1Q71L was visualized using monoclonal anti-myc antibody
coupled with Cy5 anti-mouse IgG (shown in red). Each right column is of
the same field as the left column, and arrows indicate cells
overexpressing HA-ARFs (a–d), HA-Git2-short (e and f), or
HA-Git2-short CA mutant (g and h), or both HA-Git2-short and
myc-ARF1Q71L proteins (i and j). Focuses were adjusted 3.0 μm above
the surface of the plates. Bar, 20 μm.