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. 2012 Apr 20;287(24):20443–20455. doi: 10.1074/jbc.M112.362087

FIGURE 8.

FIGURE 8.

Effect of EPS15 DN mutant on HCG-induced HLHCGR internalization and cAMP accumulation. A, HEK-HLHCGR cells coexpressing empty vector or EPS15 DN plasmid were assayed by ELISA for surface receptor levels both in the absence and in the presence of HCG (10 IU/ml, 30 min). The EPS15 DN construct decreased HCG-induced HLHCGR as compared with cells that were not transfected or were transfected with empty control vector. B, immunofluorescence analysis showing the inhibition of HCG-stimulated (10 IU/ml HCG, 30 min) HLHCGR redistribution by coexpression of EPS15 DN mutant. C, cAMP production was increased in HCG-stimulated HEK-HLHCGR cells expressing EPS15 DN mutant as compared with cells expressing either not transfected or transfected with empty vector. D, using a GST-GGA3 PBD pulldown assay, the levels of endogenous ARF6-GTP under basal conditions and in the presence of HCG (10 IU/ml, 30 min) were not affected by coexpression of EPS15 DN mutant. Total ARF6 and ARF6-GTP were visualized by immunoblot analysis with an anti-ARF6 mouse mAb. Densitometric analysis of ARF6-GTP is displayed as a histogram after normalizing to the expression of total ARF6 present in each sample.