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. 2021 Apr 27;22(9):4553. doi: 10.3390/ijms22094553

Figure 1.

Figure 1

Expression of orexin receptors and the effect of orexin A on CRH receptor and POMC expression in AtT20 cells. (A) The expression of mRNAs encoding OX1R, OX2R, and RPL19 was examined by RT−PCR in AtT20 cells compared with that in mouse gonadotrope LβT2 cells. MM: molecular weight marker. (B) After preculture, cells were treated with orexin (ORX) in serum-free media. After 24−h culture, total cellular RNA was extracted, and after 24−h or 48−h culture, cell lysates were extracted. The mRNA expression levels of Crhr1 were quantified by qPCR, and the expression levels of target genes were standardized by the RPL19 level in each sample. The cell lysates were analyzed by immunoblotting (IB) with anti−CRHR1 and anti-actin. (C) After preculture, cells were treated with ORX alone or with the combination of ORX and CRH in serum-free media. After 24−h culture, total cellular RNA was extracted, and the mRNA expression levels of POMC were quantified by qPCR. The expression levels of target genes were standardized by the RPL19 level in each sample. Results are shown as means ± SEM of data from at least three independent experiments. Statistical analysis was performed by the unpaired t−test (B) or ANOVA (B,C). * p < 0.05 vs. between the control groups.