Purification and detection of the wild type and the truncated PP13.

<p>A - Purification and characterization of heterologously expressed His-tagged truncated PP13. Expression of 6 His-tagged PP13 in <i>E. coli</i> was induced by IPTG. The protein was purified on Ni–NTA–agarose under denaturing conditions and separated on 15% SDS–PAGE. The total proteins were visualized by Gel Code blue staining. CL-clear lysate, Pel-cell pellet, Sup-Supernatant, FT-Flow through, W1 - 6 M urea, W2–4 M urea, W3 - without urea. B - The combined eluted fractions were separated by SDS-PAGE and stained by Gel-Code blue and the wildtype and truncated protein are shown. C - Proteins were electrotransferred by Western-blot. Only the His-tagged wild type (lane-1) but not the truncated PP13 (lane-2) was detected with the ELISA capture mAb (mAb1) and the detecting mAb (mAb2) as revealed with marking by HRP-conjugated rabbit anti mouse IgG and ECL detection reagent.</p

Capture ELISA.

<p>Plates were coated with the wild type PP13 or the truncated variants at 2.5 µg/ml, blocked with 1% BSA in PBS and then incubated with serial dilutions of pAbs (A) or of the 1^st, 2^nd and anti Histidine mAbs followed by development with goat ant rabbit (A) or anti mouse (B) IgG conjugated to HRP. 5A – The signal is gradually reduced over a log dilution of the antibody concentration with either the wild type (diamonds, purple) or truncated (circles, blue) PP13 variant compared to the pre-immune response (diamonds and circles, respective colors). 5B – Both first (top panel) and second mAbs (middle panel) recognized the wild type PP13 (blue) but not the truncated (red) protein. Antibodies to the His-Tag (bottom panel) recognized both of the proteins.</p

Design of the truncated PP13.

<p>Top right insert - the exon and intron organization of PP13 DNA (LGALS13 or galectin 13) showing the 5 and 3 prime ends. The 4 exons are colored black (exon 1), blue (exon 2), orange (exon 3) and brown (exon 4). Figure body - the amino acid sequence of the anticipated protein with amino acid colored according to the respective exons. Red colored – the methionine added to the his tag. The sequences of the wild type recombinant PP13 (rPP13) (top line in each couple sequences) is constructed of 164 amino acids that are aligned with the sequence of the DelT₂₂₁ (Truncated) (the lower of each couple), made of 98 amino acids. First line couple - the his-tag added to the N-terminal followed by the sequences of the first short exon (black), second exon (blue) and a part of the third exon (orange), which are identical in both molecules. Second line couple - continuation of exon 3 (orange) and beginning of exon 4 (brown) for the wild type and for the truncated variant lacking 28 amino acids of exon 3 and 7 amino acids of exon 4. Third line – the rest of exon 4 of the wild type, which is completely missing in the truncated variant (31 amino acids) and the C terminal tail, which is identical in both molecules.</p

Western blots with polyclonal pAbs.

<p>The recombinant (rPP13) wild type PP13, its truncated variant (DelT₂₂₁) and the native molecule (nPP13) purified from the placenta were separated by 15% SDS PAGE under non-reducing (A) or reducing (B) conditions, electro transferred to nitrocellulose and reacted with rabbit pAbs to PP13 (J67), pre-immune rabbit IgG (Jo) or without antibody (HRP negative control) followed by incubation with goat anti rabbit IgG conjugated with HRP. A single band is shown for the native and truncated PP13 in both reduced and non-reduced conditions. The recombinant wild type generates a single band in reduced conditions but in non-reduced conditions dimer, trimer and tetramer PP13 are revealed. The recombinant PP13 is heavier than the native protein due to the his-tag and extra tail sequence in the recombinant protein.</p

Dot blots.

<p>A – Both 1^st and 2^nd mAbs recognize the wild type PP13 over background at 10 ng/ml (top panel). Using the same conditions the 1^st mAb barely recognizes the truncated PP13 and the 2^nd mAb doe’s not show a signal at all. B – Serial concentration of the wild type and truncated PP13 variants were placed on nitrocellulose, reacted with rabbit polyclonal antibodies to PP13 (pAbs) (top panel), or non-immune serum (NRS, middle panel) or without antibodies (HRP negative control) and reacted with goat anti rabbit IgG conjugated to HRP.</p

Studies in pregnant rats.

<p>A. Mean arterial pressure (MAP) of pregnant rats after sub cutaneous implantation of slow releasing osmotic pumps with PP13 (black bars) (n = 9), the DelT₂₂₁ mutant PP13 (dark gray bars) (n = 6) or saline (light gray bars) (n = 6). Mean values ± SD of MAP are presented from day 8 (before pump implantation), and after implantation on days 10, 13 and 15 of gestation (2, 5 and 7 days after pump implantation). P values show significances between saline controls and PP13 and saline controls and DelT₂₂₁. B. The main uterine vein diameters of pregnant rats after pumps implantation is shown for 4 rats in each of the PP13 group (black bars), the DelT₂₂₁ mutant group (dark gray bars) or saline control group (light gray bars). Mean values ± SD of the main uterine veins are presented for the 21^st day of gestation (e.g. –9 days after the pumps were emptied from their releasing material). P values show significances compared to the saline control group and the DelT₂₂₁ mutant group.</p

Effects of NOS inhibition and endothelial denudation on G1 vasodilation in uterine radial arteries from pregnant rats.

<p>G1 (10⁻⁷ M) was tested on intact radial uterine arteries in absence (Control, n = 8) vs. the presence of the nitric oxide synthase inhibition using a combination of L-NNA+L-NAME (n = 5). G1 was also tested on radial uterine arteries without endothelium (Denuded, n = 5). Vasodilation is expressed as a percentage of maximal relaxation (max) in papaverine and diltiazem. Data are reported as mean ± SEM. ***p<0.001.</p

GPER expression in uterine radial arteries from nonpregnant and pregnant rats. Western blot showing.

<p>GPER protein expression in uterine arteries homogenates from non-pregnant (NP) and pregnant (P) rats. Side panel shows densitometric analysis of the blot normalized to β-tubulin. Percentage changes were evaluated as mean ± SEM of 3 experiments for each group. °p < 0.05 for the expression in P vs NP.</p

Effect of the specific GPER antagonist, G15, on G1 vasodilation in uterine radial arteries from pregnant rats.

<p>Inhibition of G1 (10^-7M; n = 8) induced vasodilation of uterine arteries from pregnant rats by the GPER-specific antagonist G15 (10^-5M, G1 ± G15, n = 5). Vasodilation is expressed as a percentage of maximal relaxation (% max) measured in a relaxing solution containing papaverine and diltiazem. Data are reported as mean ± SEM. ***p<0.001.</p

Concentration-response curves to G1 vasodilation in pressurized uterine radial arteries from non-pregnant (NP) vs. pregnant (P) rats.

<p>Uterine arteries were constricted with phenylephrine and then treated with the GPER agonist G1 at different concentration. An example of experimental records are showed in trace A and B for NP rat and in traces C and D for P rat. The Vasodilation of G1 was summarized in E and is expressed as a percentage of maximal relaxation (max) obtained in presence of papaverine and diltiazem. Data are reported as mean ± SEM; n indicates number of experiments. ***p <0.001.</p