Fig. 4
In GIST-T1 cells, PKD2, but not PI4KIIIβ, is an upstream molecule of PLD2. (a) GIST-T1 cells were transfected with the indicated siRNAs and cultured for 30 h. Lysates were immunoblotted with the indicated antibodies. pPLD2Y511, phospho-PLD2 Y511. Levels of pPLD2Y511 are expressed relative to the control cell sample after normalisation with PLD2. (b) GIST-T1 cells were treated with 200 nM imatinib (IMA, a KIT tyrosine kinase inhibitor), 10 µM CRT0066101 (CRT, a PKD inhibitor), 20 µM PIK-93 (a PI4KIIIβ inhibitor), or 20 µM CAY10594 (CAY, a PLD inhibitor) for 4 h. Cells were immunostained with anti-PI4P (green) and golgin97 (a Golgi/TGN marker, blue). Arrowheads indicate the Golgi/TGN area. Scale bars, 20 μm. (c) The graph shows the correlation coefficients (Pearson’s R) between PI4P and golgin97. Values represent mean ± SD (n = 11–22). Data were subjected to one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison post-hoc test. A representative analysis from two independent experiments is shown. ***P < 0.001, NS, not significant. Note that PI4P in the Golgi/TGN area was decreased by treatment with imatinib, CRT0066101, and PIK-93, whereas it was retained in the presence of CAY10594. (d) GIST-T1 cells were treated with 20 µM PIK-93, 10 µM CRT0066101, or 20 µM CAY10594 for 8 h. GGA1 was immunoprecipitated with anti-GGA1 antibody. The immunoprecipitates were immunoblotted for GGA1 and γ-adaptin. IP, immunoprecipitation. Immunoblots of a control antibody IP are shown in Suppl. Fig. S2f.
