The incubation conditions and the catalogue numbers for all antibodies used.

<p>The incubation conditions and the catalogue numbers for all antibodies used.</p

Western blot of protein lysate from PBMCs, amniocytes, myocytes.

<p>50 µg of whole cell lysate from PBMCs, amniocytes, myocytes were subject to western analysis. Membranes were probed with the primary antibody -SC-23092 to detect a Mr∼34 000 product (<b>A</b>; Lanes 1–3). The secondary antibody control blot is also shown (<b>B</b>; Lanes 1–3). β-actin was used as a loading control. Lane 1 = PBMCs, lane 2 = amniocytes, lane 3 = myocytes. Multiple bands are seen on the blot. A band appears at Mr∼34 000 in amniocytes, faintly in the myocyte lane, but is absent in the positive control PBMCs lane. However, the strongest bands appear at Mr∼15 000 and at just above Mr∼43 000 in all lanes. Myocytes from passage zero, one, two, three and four (lanes 1–5 respectively) were also examined for CRTH2 expression showing a very faint band at Mr∼34 000, with no effect of passage number (<b>C</b>).</p

CRTH2 mRNA is expressed in amniocytes and myocytes.

<p>mRNA was isolated from cultured amniocytes, myocytes, PBMCs, choriodecidual and placental extracts and converted to cDNA (n = 6). Qualitative PCR was used with three primer sets to amplify CRTH2 showing product sizes of 309 bp, 265 bp, and 114 bp. Non-template and reverse transcriptase negative controls were used and mRNA from placenta, choriodecidua and peripheral blood mononuclear cells were used as a positive controls. (<b>A</b>,<b>B</b>): Non-template control (lane 1), amniocytes (lane 2), choriodecidua (lane 3), myocytes (lane 4), placenta (lane 5); (<b>C</b>): Reverse transcriptase controls (lanes 1,3,5,7,9), PBMCs (lane 2), amniocytes (lanes 4,6), myocytes (lanes 8,10).</p

Pyl A has no effect on NF-κB p65 activity in amniocytes and myocytes.

<p>Protein was extracted from IL-1β stimulated and Pyl treated cells and levels of nuclear p65 and phosphorylated p65 (p-p65) were examined using immunoblotting. A dose response of 0.1–32 µM of Pyl A was used. Representative immunoblots are shown for amniocytes, (<b>A</b>) and myocytes (<b>B</b>). Immunoblots were re-probed for β-actin as an internal loading control. Densitometric analysis of the immunoblots was conducted revealing no effect of Pyl A on p65 or p-p65 in amniocytes (<b>C</b>, <b>E</b>) or myocytes (<b>D</b>, <b>F</b>). NS = non-stimulated (non-IL-1β treated cells). Effect of treatment was examined for statistical significance using ANOVA of repeated measures with Bonferroni’s multiple comparison test; *<i>P</i><0.05.</p

An overview of the mean fluorescence intensities and percentages in the gated cells during detection of endogenous CRTH2 in lymphocytes, amniocytes and myocytes.

<p>An overview of the mean fluorescence intensities and percentages in the gated cells during detection of endogenous CRTH2 in lymphocytes, amniocytes and myocytes.</p

The primer sequences used for amplification of CRTH2.

<p>The primer sequences used for amplification of CRTH2.</p

Flow cytometry for the detection of endogenous CRTH2.

<p>Lymphocytes were gated according to forward scatter and side scatter and T helper cells were identified using CD4 as a cell surface marker (n = 6). A representative cytogram is presented with the right upper quadrant showing CRTH2⁺/CD4⁺ lymphocytes (<b>A</b>). Histograms showing no staining, isotype control and CRTH2⁺ labelled lymphocytes (<b>B</b>), amniocytes (<b>C</b>) and myocytes (<b>D</b>), (n = 5).</p

15dPGJ2 reduced NF-κB p65 activity in amniocytes and myocytes.

<p>Protein was extracted from IL-1β stimulated and 15dPGJ2 treated cells and levels of nuclear p65 were examined using immunoblotting. A dose response of 0.1–32 µM of 15dPGJ2 was used (n = 3). Representative immunoblots are shown for amniocytes, (<b>A</b>), myocytes (<b>B</b>). Immunoblots were re-probed for β-actin as an internal loading control.</p

Detection of radiolabelled CRTH2 in pSG5 expression vector by x-ray and immunoblot.

<p>The in vitro transcription translation kit was used with 35^S Methionine to demonstrate the expression of CRTH2 and to provide a positive control for detection of CRTH2. A Mr∼34 000 product was detected by x-ray (Lane 2). The progesterone expression vector was used as a control for the TNT kit (<b>A</b>). The protein lysate from the TNT kit was subjected to western analysis; Negative Control: TNT kit with no plasmid DNA (lane 1), Negative control: Progesterone PSG5 Expression vector (lane 2), Positive control: 35^S Methionine labelled CRTH2 (Lane 3), Cold Methionine CRTH2 protein product (lane 4). Membranes were probed with 3 commercial antibodies; SC-23092 (<b>B</b>), SC-21798 (<b>C</b>), ProSci 4027 (<b>D</b>). None of the antibodies detected CRTH2.</p

The effect of 15dPGJ2 on basal NF-κB p65 phosphorylation in peripheral blood mononuclear cells.

<p>PBMCs were treated with 32 µM of 15dPGJ2 for 2 hours with or without preincubation with 2 µM of GSKCRTH2X for 45 mins. Whole cell protein lysate was examined for levels of phosphorylated p65 (p-p65) using immunoblotting. A representative immunoblot from n = 6 samples are shown (<b>A</b>). Immunoblots were reprobed for β-actin as an internal loading control. Densitometric analysis of the immunoblots was conducted revealing a complete inhibition of p-p65 levels with 15dPGJ2. Pre-incubation with the CRTH2 antagonist GSKCRTH2X had no effect on inhibition (<b>B</b>). J2 = 15dPGJ2, GSKX = GSKCRTH2X. Effect of treatment was examined for statistical significance using ANOVA of repeated measures with Bonferroni’s multiple comparison test; **** <i>P</i><0.0001.</p