Figure 2.

Acidosis-induced ER stress response is augmented by overexpression of G protein-coupled receptor 4 (GPR4), but not the signaling defective GPR4 mutant, in HUVEC. (A–D) HUVECs transduced with the control vector (Vector), GPR4 expression construct (GPR4), or GPR4 R115A mutant expression vector (GPR4 R115A) were treated in EGM-2/HEM buffered media at physiological (pH 7.4) or acidic (pH 6.4) conditions for 0.5–1 h (p-IRE1α) or 5 h (ATF3, ATF4, and ATF6). Cell lysates were then collected and separated by electrophoresis. Protein expression of (A) ATF3, (B) ATF4, (C) active/cleaved ATF6, and (D) phosphorylated-IRE1α was detected using the specific antibodies. β-Actin or TATA-binding protein (TBP) expression was used as a loading control. The arrow indicates the target band. After being normalized to the loading control, the target bands were quantified by densitometry using the ImageJ software (version 1.51, National Institutes of Health, Bethesda, USA). The relative protein expression level at pH 7.4 of HUVEC/Vector was set as 1.0-fold. (E) HUVECs, as used in (A–D), were treated at pH 8.4, 7.4, or 6.4 for 5 h. Total RNA was isolated and cDNA was synthesized. Spliced and unspliced XBP-1 mRNA isoforms were examined by RT-PCR. The results shown are representative of at least two biological repeats.