Figure 4.

Acidosis-induced ER stress response is attenuated by a GPR4 small molecule inhibitor in HUVEC. HUVEC/Vector cells were treated for 0.5–1 h (p-IRE1α) or 5 h (ATF3, ATF4 and ATF6) with EGM-2/HEM pH 7.4 and 6.4 media or with pH 6.4 media containing 10 and 50 µM of the GPR4 inhibitor, for which one-hour pretreatment in EGM-2 medium with the same concentrations of GPR4 inhibitor was performed. Cell lysates were then collected and separated by electrophoresis. Protein expression of ATF3, ATF4, active/cleaved ATF6, and phosphorylated-IRE1α was detected using the specific antibodies. β-actin or GAPDH 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, MD, USA). The relative protein expression level at pH 7.4 of HUVEC/Vector was set as 1.0-fold. The results shown are representative of two or more biological repeats.