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. 2011 Oct 4;22(4):661–676. doi: 10.1038/cr.2011.161

Figure 1.

Figure 1

PAQR10 and PAQR11 are localized in the Golgi apparatus and activate Ras/ERK signaling. (A) Ectopically expressed PAQR10 and PAQR11 are localized in the Golgi apparatus. Hela cells were transfected with Flag-tagged PAQR10 and PAQR11, followed by immunostaining and confocal analysis. The Golgi was labeled by Golgin-97. (B) Endogenous PAQR11 is localized in the Golgi. A549 cells were stained with affinity-purified, anti-PAQR11 antibody and the Golgi marker Golgin-97. (C) Dose-dependent activation of ERK phosphorylation by PAQR10 and PAQR11. HEK293T cells were transiently transfected with increasing amounts of Flag-tagged PAQR10 (left panel) or Flag-tagged PAQR11 (right panel). Twenty-four hours after transfection, the cells were serum-starved for 6 h and the cell lysate was used in immunoblotting, using antibodies against phosphorylated ERK, total ERK and Flag tag. The same experiment was carried out at least three times, and one of the representative data is shown. This is the same for most immunoblotting analyses throughout the study. (D) PAQR10 and PAQR11 enhance and prolong EGF-induced ERK signaling. HEK293T cells were transiently transfected with Flag-tagged PAQR10 and PAQR11. Twenty-four hours after transfection, cells were serum-starved for 6 h before treatment with EGF (100 ng/ml) for various lengths of time. Total cell lysate was used in immunoblotting. (E) PAQR10 stimulates expression of transcription factors downstream of ERK in a dose-dependent manner. HEK293T cells were transiently transfected with increasing amounts of Flag-PAQR10. Twenty-four hours after transfection, the cells were collected and real-time RT-PCR was used to determine the expression of cFos, junB, Egr-1, and actin. Fold change of each gene compared with actin was shown as mean ± SD for three independent experiments. The fold change of control group was set to 1. *P < 0.05 and **P < 0.01 in comparison with the control group. (F) PAQR10-mediated stimulation of ERK phosphorylation is inhibited by GFP-RBD and PD98059. HEK293T cells were transiently transfected with the plasmids or treated with 10 μM of PD98059 as indicated. Twenty-four hours after transfection, the cells were serum-starved for 6 h, and the total cell lysate was used in immunoblotting. The right panel represents pERK/total ERK ratio (mean ± SD) calculated from densitometer analyses of three independent experiments. **P < 0.01 as compared to the control group and ^^P < 0.01 as compared to PAQR10 alone.