Quantitative analysis of the
distribution of ARF-GEFs relative to Golgi markers and coat
proteins. The extent of overlap observed in the perinuclear
area by using antibodies to the indicated proteins was
measured as described in MATERIALS AND METHODS. The
percentage of IF signal intensity present in shared pixels
(yellow) relative to total signal was measured for both the
red (top) and green (bottom) channels. Red and green only
values were defined as the difference between 100% and the
overlap measured for the corresponding channel. Red signal
was obtained by staining with polyclonal anti-GBF1 or anti-
BIG1, whereas green signal was obtained with monoclonal
anti-p115, anti-β-COP, or Alexa488-conjugated polyclonal
anti-BIG1 (see legends of Figures 2, 3, and 8 for details).
The number of separate images used for quantitation was
five for GBF1/p115, four for TGN38/BIG1, five for
GBF1/BIG1, six for GBF1/β-COP, and six for BIG1/β-COP.
Error bars correspond to the SD of the mean.