Representative microphotographs of immunohistochemical (IHC) localization of RXFP1 in the canine CL at selected time points of pregnancy.

<p>Pre-implantation (A), post-implantation (B), mid-gestation (C) and at prepartum luteolysis (D). RXFP1 is localized to the luteal cells (open arrows in (A-D)), endothelial cells (solid arrows in A and C) and macrophages (open arrowheads in (A-D)). There is no background staining in the isotype control (insert to (D)).</p

Representative microphotographs of immunohistochemical (IHC) localization of RLN in the canine CL at selected time points of pregnancy.

<p>Pre-implantation (A), post-implantation (B), mid-gestation (C) and at prepartum luteolysis (D). RLN is localized to the lutein cells (open arrows in (A–D)). There is no background staining in the isotype control (insert to (D)).</p

Isolation of hypophysis and adehohypophyseal expression of RLN-system.

<p>Representative photographs of opened skull of a dog before (A, dorsal view and B, dorsocaudal view), and after (C, dorsocaudal view) removal of the brain including the hypophysis (D). The hypophysis was localized in the saddle-shaped depression of the sphenoid bone, so-called Turkish saddle (<i>Sella turcica</i>). Proximally, directly above the pituitary gland, the optic chiasm can be localized. Parts of the optic chiasm are attached to the isolated hypophysis (D). E) Expression of <i>RLN</i>, <i>RXFP1</i>, <i>RXFP2</i> and <i>GAPDH</i> in canine adenohypophysis by conventional, qualitative PCR. The mRNA of all RLN system members was detectable, with <i>RLN</i> and <i>RXFP2</i> signals appearing weaker than those for <i>RXFP1</i>.</p

Expression of (A) <i>RLN</i>, (B) <i>RXFP1</i> and (C) <i>RXFP2</i> in canine CL at selected time points of pregnancy.

<p>Data are presented as geometric means Xg ± SD. (★) = P<0.01, (★★) = P<0.001.</p

Hematoxylin-eosin (HE) staining (A, D) and immunolocalization of PRL (B, E) and RLN system (C, F, G) in canine adenohypophysis (two series of consecutive sections are presented: A-C and D-G).

<p>HE staining was performed in order to distinguish basophilic (solid arrows in A and D) and acidophilic hypophyseal cells (open arrows in A and D). The latter include PRL-secreting cells, so-called lactotrophs, identified additionally by IHC with anti-PRL antibody (open arrows in B and E). RLN signals were observed in lactotrophs (open arrows in C) and in other hypophyseal cells, which remained negative for PRL (solid arrows in C). RXFP1 and RXFP2 stained extensively in hypophyseal cells with signals appearing more prominent for RXFP1 than RXFP2 (open arrows in F and G, respectively). There is no background staining in the isotype controls (H, I, J, K).</p

Sedimentation analysis of <i>Neq</i>SSB-like (A) and standard proteins (B).

<p>The proteins were analyzed on a 12% polyacrylamide gel. Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), with the molecular mass of proteins marked. Lane 1–19: fraction number. 50 μl of 150 μM <i>Neq</i>SSB-like and the corresponding amounts of standard proteins were centrifuged in linear 15 to 30% (w/v) glycerol gradients, as described in the “Methods”. The fractions with proteins were analyzed by SDS-PAGE. The fractions at which the maximal amount of protein appears are shown by arrows in each panel. The standard proteins used are: L, lysozyme (14 kDa); CA, carbonic anhydrase (29 kDa); BSA, bovine serum albumin (66 kDa) and AD, alcohol dehydrogenase (150 kDa). The oligomerization state estimation of <i>Neq</i>SSB-like was made with these proteins.</p

Binding of <i>Neq</i>SSB-like to M13 ssDNA.

<p>Lanes 1–8 contain (0.07 pmol) of M13 ssDNA and 0, 3.5, 7, 14, 28, 56, 112 and 224 pmoles of <i>Neq</i>SSB-like, respectively.</p

The determination of ssDNA-binding activity half-life, using gel mobility shift assays, for <i>Neq</i>SSB-like and <i>Taq</i>SSB.

<p><i>Neq</i>SSB-like represented by the open circles and <i>Taq</i>SSB, shown as open triangles.</p

The expression and purification of <i>Neq</i>SSB-like from <i>E</i>. <i>coli</i> TOP10F’+pBAD/NeqSSBHT.

<p>The proteins were analyzed on a 12% polyacrylamide gel. Lane M: Unstained Protein Weight Marker (Fermentas, Lithuania), with the molecular mass of proteins marked. Lane 1: soluble protein cell extracts after arabinose induction of protein expression (10 μl). Lane 2: <i>Neq</i>SSB-like after the Ni²⁺-affinity chromatography step (10 μl). Lane 3: <i>Neq</i>SSB-like after His-tag cleavage with TEV protease (10 μl). Lane 4: <i>Neq</i>SSB-like after chromatography on an ssDNA-cellulose column (10 μl).</p