Figure 2. Membrane trafficking of KCC4.

(A) EGF and IGF-1 stimulate the membrane recruitment of KCC4. Ovarian cancer OVCAR-3 cells were incubated in the absence (control group) or presence of EGF (100 ng/ml) or IGF-1 (100 ng/ml) for 30 min. Upper panel: representative pictures from 6 different experiments. Lower panel: the quantitative analyses of KCC4 membrane trafficking in response to growth-factor stimulation. Each column represents mean ± S.E.M. of at least 250 cells. *P<0.01 by unpaired t test. N:nucleus. Scale bar, 10 μm. (B) IGF-1 increases KCC4 surface expression, detected by surface biotinylation assay. Total cell lysates were prepared in parallel for comparison. Akt: a marker of cytosolic protein; E-cadherin: an internal control of membrane protein. Left panel: a representative immunoblot. Right panel: the densitometric quantification of KCC4 surface expression. Each column represents mean ± S.E.M. (n=3). *P<0.01 by paired t test (C) & (D) IGF-1 stimulates KCC4 redistribution between ER and Golgi. (C) Representative confocal images of ovarian cancer OVCAR-3 cells incubated with or without 100 ng/ml IGF-1 for 30 min. Calnexin: ER marker protein; Man II: mannosidase II, Golgi marker protein. Scale bar, 10 μm. (D) The ratio of mean fluorescence intensity of KCC4 in the Golgi (F_Golgi) over its mean fluorescence intensity in the ER (F_ER) was calculated at different time points and used as a measurement of KCC4 redistribution. Each points represents mean ± S.E.M. of at least 60 cells. *P<0.01 by paired t test