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. 2017 Jan 27;18(2):278. doi: 10.3390/ijms18020278

Figure 3.

Figure 3

Acidosis-induced ER stress response is alleviated by knocking down GPR4 expression in HUVEC. HUVECs transduced with the control shRNA (ctrl shRNA) or GPR4 shRNA (GPR4 shRNA) 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-eIF2α), 1 h (XBP-1), or 5 h (ATF3 and ATF6). (A–C) Cell lysates were collected and separated by electrophoresis. Protein expression of (A) phosphorylated-eIF2α, (B) ATF3, and (C) active/cleaved ATF6 was detected using the specific antibodies. β-Actin expression was used as a loading control. 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/Control-shRNA was set as 1.0-fold. (D) Total RNA was isolated from the cells and cDNA was synthesized. Spliced and unspliced XBP-1 isoforms were examined by RT-PCR. The results shown are representative of two or more biological repeats.