Fig. 7. PACAP regulates Otp post-transcriptionally. (A,B) Western blot of equal amounts of protein extracts from HEK293 cells with an anti-Otp antibody followed by an anti-β-actin antibody that served as an internal reference. (A) HEK293 cells were transiently transfected with different combinations of zebrafish cDNAs as indicated and harvested 24 hours post transfection. pac1^* encoded a constitutively activated form of pac1. The full-length form of zebrafish pacap1b precursor gene, gives rise to several proteolytically cleaved neuropeptides including growth hormone releasing hormone (GHRH) and PACAP1b (Wang et al., 2003). 1-5pacap1b cDNA had an internal deletion of the first five amino acids of the mature PACAP peptide. (B) HEK293 cells were transfected with the indicated zebrafish cDNAs and 24 hours later monolayer cultures were treated with different concentration of a synthetic PACAP₃₈ peptide for a period of 120 minutes. (C,D) Western blot of nuclear-enriched protein extracts from PC12 cells using either an anti-Otp or anti-Rcc1 antibody (internal control for a ubiquitous nuclear protein). Cells were treated with different concentrations of either PACAP₃₈ peptide alone (C), 50 µg/ml cycloheximide (Control+CHX) or PACAP₃₈+CHX (D) and harvested at the indicated time points. (E) Standardized Otp protein and RNA (n=6) levels as a function of time after PACAP₃₈ application to PC12 cells. otp RNA levels were determined by quantitative real-time PCR (RNA at 10 nM). (F) Kinetics of newly synthesized Otp protein in the absence (Control) or presence of 10nM PACAP. PC12 cells were incubated with methionine-free medium containing [³⁵S]methionine (at 100 µCi/ml). Protein extracts were made at the indicated times and equal amounts of total protein were subjected to immunoprecipitation with an anti-Otp antibody followed by gel electrophoresis and autoradiography (inset). The correct position of the Otp band was confirmed by performing immunoprecipitation and western blot on the same extracts and by Otp peptide displacement. Ab, antibody; CHX, cycloheximide; IB, immunoblot.