Fig. 2
CAY10594-induced reduction of KIT is due to lysosomal degradation. (a, b) GIST-T1 cells were treated with 20 µM CAY10594 (CAY, a PLD inhibitor) and/or 20 mM NH4Cl (inhibits lysosomal proteases) for (a) 16 h or (b) 4 h. (a) Lysates were immunoblotted for KIT, phospho-KIT Y703 (pKITY703), transferrin receptor (TfR, a recycling endosome protein), and β-actin. Relative band intensities normalised with each control sample are shown. The upper bands of KIT were quantified. (b) Cells were immunostained with KIT (green) and LAMP1 (lysosome-associated membrane protein 1, red). Scale bars, 20 μm. Note that KIT was found in lysosomes in GIST cells treated with CAY10594 plus NH4Cl. (c) The graph shows the correlation coefficients (Pearson’s R) between KIT and LAMP1. Values represent mean ± SD (n = 15–29). A representative analysis from two independent experiments is shown. *P < 0.05, ***P < 0.001, Student’s t-test.
