Prolactin stimulation of CID-9 cells increases PAD3 mRNA and protein expression.

<p>Equal numbers of CID-9 cells were grown in phenol red free media with charcoal-stripped FBS overnight. The following morning cells were treated with vehicle or 5 μg/mL of prolactin for 24 or 48 hours. (A) Prolactin treatment significantly increases PAD3 mRNA. Total RNA was extracted from CID-9 cells, reverse transcribed, and resulting cDNA examined by qPCR with intron spanning primers specific for PAD3 or GAPDH as the reference gene control. All data values were normalized to vehicle treated control and data are presented as means ± SEM. Means were separated using SNK ANOVA and * indicates significant difference (P<0.05). (B) Prolactin treatment increases PAD3 protein levels. The top panel shows a representative western blot, while the graph in the bottom panel represents the quantification of western blots using BioRad Image Lab 4.0 (n = 4, P<0.05). Protein concentrations of cell lysates were determined by BCA assay and equal amounts loaded and examined by western blot. Membranes were probed with a rabbit anti-PAD3 antibody or with anti-β-actin as a loading control. The positive control was generated by overexpressing human PAD3 in CID-9 cells.