Time-course of [Ca²⁺]_c produced by progesterone and/or peptide 1932.

<p>The figure illustrates the result of a typical experiment. Cells were suspended in HBSS buffer with no calcium. Peptide (1700 nM) was added two minute before progesterone (10 μM). After 250 s calcium was added to reach a concentrations of 1 mM. Inset: Maximal increase of [Ca²⁺]_c, reported as Δ[Ca²⁺]_c, after the addition of increasing amounts of progesterone. The results shown correspond to the means± SEM of 10 experiments ANOVA. Effects due to progesterone in the absence of calcium: p<0.0001 and in the presence of 1 mM calcium: not significant (not shown in the inset). <i>Post-hoc</i>: Scheffè’s test at p level 0.05. The asterisks shown statistical significance.</p

Inhibition experiments with AG205 and p1932 peptide.

<p>The experiments were carried out on PE/CA PJ15 labeled with FURA-2 AM. After 50 seconds in the incubation medium, P4 (5 μM, Fig 6A; 10 μM, Fig 6B) was added and the increase in cytosolic calcium as Δ[Ca²⁺]c measured. The PGRMC1 inhibitor AG 205 (0.01–1.0 μM) and/or p1932 peptide (0.01–1.0 μM), was added in the medium 50 seconds prior to 5 or 10 μM progesterone. In the presence of AG205 or p1932 peptide, the effect of P4 (5–10 μM) on Δ[Ca²⁺]c was statistically inhibited in a dose dependent manner. When AG 205 (0.01–1.0 μM) and p1932 peptide (0.01–1.0 μM) were added in combination, the observed inhibitory effect was higher than in samples treated with either agent alone. Each data correspond to the mean± SEM of six independent measurements.</p

PGRMC1 identified by LC-ESI-MS/MS.

<p>Listed are the following protein parameters: alphanumeric unique protein sequence identifier (Accession), protein identifier characters with a naming convention (Entry name), Gene name and protein name (Description) provided by UniProtKB protein knowledgebase; the calculated molecular weight of the protein in kilo-Dalton units (MW), the theoretically calculated isoelectric point (cal. pI) and the protein number of amino acids (# AAs); protein identification’s SEQUEST HT Score; percentage of protein sequence covered by identified peptides (Cov); number of peptides unique to the protein (# Unique Peptides); number of the identified peptides matching to the protein (# Peptides); total number of identified peptide sequences (peptide spectrum matches) (# PSMs) obtained by two different experiment (Exp 1 and Exp 2). In the table are listed the PGRMC1 peptides following parameters: the identified amino acidic peptide Sequence; Charge state of the precursor ion; the theoretical protonated monoisotopic peptide mass, in daltons (MH+); mass-to-charge ratio (m/z) of the precursor ion, in daltons; the difference between the theoretical mass of the peptide and the experimental mass of the precursor ion in parts per million (ΔM); retention time of the precursor ion, in minutes (R.T.); the SEQUEST HT cross-correlation score for all candidate peptides queried from the database (Xcorr); number of Missed Cleavages.</p

Effects of 1932 salivary peptide and its fragments on [Ca²⁺]c increase produced by progesterone.

<p>After the addition of either peptide or its fragments at different concentrations (17–1700 nM), 5 μM progesterone was also added to calcium-free tissue culture medium. Data (reported as Δ[Ca²⁺]c), report the means± SEM of 10 experiments. ANOVA: Effects of peptide: p<0.001 and of concentration: p<0.001. Post-hoc: (scheffée p = 0.05). Homogeneous peptide subsets: 1. A (p1932), B (MER 1–17) and D (MER 12–20); 2. C (MER 1–17 des Pro), E (MER 1–11) and F (MER 1–8).</p

Time-course of [Ca²⁺]c produced by progesterone.

<p>The figure illustrates the result of a typical experiment. Cells were suspended in HBSS buffer with no calcium. After 250 seconds calcium was added to reach a concentrations of 1 mM. The experiment was repeated 10 times and a similar behavior was always observed. Inset: Maximal increase of [Ca²⁺]c, reported as Δ[Ca²⁺]c, after the addition of increasing amounts of progesterone. The results shown correspond to the means± SEM of 10 experiments. ANOVA. Effects due to progesterone in the absence of calcium: p<0.0001, and in the presence of 1 mM calcium: not significant (not shown in the inset). Post-hoc: Scheffè’s test at p level 0.05. The asterisks shown statistical significance.</p

Sequence of human PGRCM1 from Uniprot (O00264).

<p>In bold are evidenced the two putative consensus sequences potential target of SH3 domains as obtained by SH3-Hunter web site [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147925#pone.0147925.ref050" target="_blank">50</a>].</p