Quantitative genetics of plumage color

This is an excel file with 5 worksheets containing datasets used for different analyses shown in the paper. 1) Comparing color between sexes and developmental stages, 2) Frequency of extra-pair young (EPY) across years, 3) Comparing color of recruited vs. non-recruited individuals, 4) Within-individual color comparison of recruited individuals, and 5) data used in the animal model analysis. See the ReadMe file for more details

Pedigree3_070114

A text file containing pedigree that includes the genetic parent(s) of the nestlings that was used to determine the additive genetic variance component for calculating heritability

CORT3_070114

Text file containing all pertinent data for the bivariate animal model we ran to assess heritability of variation in baseline and stress-induced corticosterone (CORT) concentrations in nestling barn swallows. This file is similar to the excel file available in this datafile but in a format more easily accessible to R statistical program

NestlingBarnSwallowCORT2010-HeritabilityMS

Contains data for USGS-banded nestling North American barn swallows (Hirundo rustica erythrogaster) collected at day 12 between May and July 2010. Data includes site and nest location, date banded, brood size manipulation treatment, age at data collection, sex, brood number (based on breeding adult pair), clutch size, brood size, mass, presence of mites, DNA extraction number, social and genetic assignments of parents, date and time information for baseline and stress-induced corticosterone (CORT) samples, CORT concentrations, and plumage color score

DRYAD ISRAEL + COLORADO distn of paternity outcomes

This file contains the paternity outcomes associated with the experimental manipulation of phenotype in two different study locations: Israel and Colorado, US

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.</p

PAD3 is the most highly expressed PAD isoform in mouse mammary epithelial CID-9 cells.

<p>PAD3 mRNA levels are significantly higher in CID-9 cells compared to all other PAD isoforms. Total RNA was extracted from CID-9 cells, reverse transcribed, and resulting cDNA examined by qPCR with intron spanning primers specific for PAD1, PAD2, PAD3, PAD4 or GAPDH as the reference gene control. All data values were normalized to PAD1 to yield fold change, and data are expressed as means ± SEM. Means were separated using Tukey’s test ANOVA and letters indicate significant differences (P< 0.05).</p

Inhibition of PAD3 decreases the level of citrullinated proteins in CID-9 cells.

<p>CID-9 cells were treated with vehicle or 50 μM Cl4-amidine overnight. The following morning cells were lysed and equal concentrations of protein lysates examined by western blot following the AMC protocol. The positive control is <i>in vitro</i> citrullinated bulk histones.</p

Prolactin stimulates the JAK2/STAT5 signaling pathway to upregulate PAD3 expression in CID-9 cells.

<p>(A) JAK2 mediates prolactin induced PAD3 upregulation. CID-9 cells were pre-treated for 4 hours with DMSO or 3 μM JAK2 inhibitor (SD-1029). Following pre-treatment, cells were stimulated with vehicle or 5 μg/mL of prolactin for 48 hours. After the first 24 hours, cells received fresh inhibitor. Protein concentrations of cell lysates were determined by BCA assay and equal amounts examined by western blot. Membranes were probed with anti-PAD3, p-JAK2, total JAK2 antibodies or with anti-β-actin as a loading control. (B) STAT5 mediates prolactin induced PAD3 upregulation. CID-9 cells were infected overnight with 10 MOI of an adenovirus expressing GFP (Ad-GFP) or an adenovirus expressing a dominant-negative form of STAT5 (Ad-DN-STAT5) as indicated. The following morning cells were treated with vehicle or 5 μg/mL of prolactin for 24 or 48 hours. Protein concentrations of cell lysates were determined by BCA assay and equal amounts examined by western blot. Membranes were probed with anti-PAD3, GFP, p-STAT5, total STAT5 or β-actin antibodies.</p

The human <i>PAD3</i> gene promoter is prolactin responsive in CID-9 cells -276/+41 and -94/+41 base pairs of the human <i>PAD3</i> gene promoter were cloned from genomic DNA and fused to the cDNA of luciferase.

<p>CID-9 cells were transfected overnight with hPAD3–276/+41-Luc, hPAD3–94/+41-Luc, pGL3 empty, and CMV-β-galactosidase. The next morning cells were treated with vehicle or 5 μg/mL of prolactin for 24 hours. Cellular lysates were assayed for luciferase and β-galactosidase activity. Luciferase values were corrected for β-galactosidase activity, and data are expressed as adjusted light units (top panel A) or fold change in adjusted luciferase activity (bottom panel B). The inset figure in top panel A illustrates that the hPAD3–94/+41-Luc construct has significant transcriptional activity compared to the pGL3 empty. Values represent the mean ± SEM. Means were separated using Student’s T-Test with * designating significant differences with treatment (P<0.05).</p