Fig. 3.

Expression of prepro-orexin and OX1R mRNAs and the proteins in human normal and hyperplastic prostates (A and B) and expression of OX1R in PNT1A cultured cells (C). (A) RT-PCR analysis. Lane 1, DNA ladder; lane 2, prepro-orexin and OX1R mRNA transcripts from whole rat brain (positive control); lanes 3 and 4, prepro-orexin and OX1R mRNA transcripts from normal and hyperplastic prostate samples, respectively; lane 5, negative control (no cDNA input). The bottom of (A) reports the beta-actin mRNA transcripts (internal control). (B) Western blotting analysis. Lane 1, homogenates from whole rat brain (positive control); lanes 2 and 3, homogenates from normal and hyperplastic prostate tissue, respectively; lane 4, prostate homogenates treated with the antisera directed against prepro-orexin or OX1R pre-absorbed with their respective control peptides (negative control). (C) Western blotting analysis. Lane 1, homogenates from whole rat brain (positive control); lane 2, homogenates from PNT1A cell lysate; lane 3, cell lysates treated with the antiserum directed against OX1R pre-absorbed with its control peptide (negative control). The upper blots of (B) and (C) were stripped and re-probed with an anti-tubulin monoclonal antibody to ensure equal loading of proteins in all lanes (lower blots). Molecular mass markers are indicated on the left of the Western blotting panels. Similar results were obtained from four separate experiments of identical design.