Acidosis/GPR4 and Endothelial Cell Adhesion 100, 0.25% Na-deoxycholate, 1 mM EDTA, 1 mM phenyl- methylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, 5% protease cocktail inhibitor from Sigma) for 15 min at 4uC. The cell lysates were clarified by centrifugation at 14,0006 g for 15 min at 4uC. Protein concentration of the supernatant was determined by the Bradford protein assay (Bio-Rad, Hercules, CA). The expression of myc-tagged N-Epac was analyzed by Western blotting with the mouse monoclonal anti-c-myc antibody (Roche), followed by the incubation with the horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) secondary antibody (Bio-Rad). Chemiluminescence signals were detected using the Amersham ECL Advance Western blotting detection kit (GE Healthcare). Immunofluorescence The immunostaining was performed as previously described with minor modifications [15,45]. All staining steps were carried Figure 1. Acidosis/GPR4-induced adhesion of HUVEC cells. (A) HUVECs stably expressing the MSCV-huGPR4-IRES-GFP construct (GPR4) or the MSCV-IRES-GFP control vector (Vector) were grown as a flat monolayer and treated with EGM-2/HEM media at varying pH for either 5 h or 15 h (overnight) which showed similar results. Then, 66104 cells/well of U937 monocytic cells were added to adhere with HUVECs for 1 h at pH 7.4, and the static cell adhesion assay was performed as described in the ‘‘Materials and Methods’’. After the adhesion assay, the attached U937 cells were readily detected as small, round and reflectile cells under an inverted microscope with a 106 objective. Arrows indicate attached U937 cells. (B) HUVECs stably overexpressing GPR4 or the control vector were treated with different pHs as indicated. The cell adhesion assay was then performed. *, P,0.05; ***, P,0.001; compared with the pH 8.2 group. (C) HUVECs were transduced with various genetic constructs as indicated. Total RNA was isolated and mRNA levels of GPR4 were determined by real-time RT-PCR. Values were normalized to the housekeeping gene GAPDH. The expression level of GPR4 in HUVECs with the control vector was set as 1. (D) HUVECs stably overexpressing the mouse GPR4-GFP fusion gene were treated with different pHs as indicated, and then the cell adhesion assay was performed. Error bars are the mean 6 SEM. *, P,0.05, compared with pH 8.4. (E) HUVECs were transduced with two GPR4 miRNAs or control miRNA, followed by pH treatment, and then the cell adhesion assay was performed. *, P,0.05; **, P,0.01; compared with the control miRNA group at pH 6.4. (F) HUVEC/GPR4 cells were treated with different pHs for various lengths of time as indicated, and then the cell adhesion assay was performed. **, P,0.01; ***, P,0.001; compared with the 1 h group at pH 6.4. The results are depicted as the mean 6 SEM and representative of more than two independent experiments. doi:10.1371/journal.pone.0027586.g001 PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27586