Journal: Molecular and cellular endocrinology

Article Title: Oxysterol-Related-Binding-Protein Related Protein-2 (ORP2) Regulates Cortisol Biosynthesis and Cholesterol Homeostasis

doi: 10.1016/j.mce.2016.03.006

Figure Lengend Snippet: (A) Whole cell lysates were harvested from wild type, scrambled negative shRNA control, ORP2 shRNA#1 and ORP shRNA#2 cells and analyzed by SDS-PAGE and western blotting using antibodies against ORP2 (top) and GAPDH (bottom). Shown is a representative blot from experiments performed on at least 6 different occasions, each time in at least duplicate. (B) Densitometric analysis of protein isolated from wild type, scrambled shRNA, ORP2 shRNA#1, and ORP2 shRNA#2, cells that were subjected to western blotting. The expression of ORP2 protein is normalized to GAPDH and the data graphed represent the mean ± SEM of five separate experiments, each performed in at least duplicate. (C) RNA from untreated wild type, scrambled shRNA, ORP2 shRNA#1, or ORP2 shRNA#2 was isolated for analysis of ORP2 expression by real time RT-PCR. Data are graphed as fold change in mRNA expression over wild type and normalized to the mRNA expression of β-actin and represent the mean ± SEM of three separate experiments, each performed in triplicate. (D) RNA isolated from wild type, scrambled and ORP2 shRNA#1 was subjected to microarray analysis. The graph data represent multilevel gene ontology (GO) analysis of the biological processes that were significantly changed in ORP2kd cells when compared to the wild type. The vertical axis represents the GO terminology and the x-axis represents the proportion of genes within each category that were significantly changed.

Article Snippet: Microarray analysis was carried out by Phalanx Biotech Group, Inc. (Palo Alto, CA) using the Human Whole Genome OneArrayTM DNA Microarray (HOA_004) on RNA isolated from wild type, scrambled shRNA, and ORP2 shRNA#1 cell lines.

Techniques: shRNA, Control, SDS Page, Western Blot, Isolation, Expressing, Quantitative RT-PCR, Microarray