SmAV protein content increases with gestation in both the human and the rat.

<p>A. Specificity of the anti SmAV antibody. B. Gestation-dependent increase in SmAV expression in human myometrium. Densitometry analysis of smooth muscle Archvillin (SmAV) protein levels from immunoblots of human myometrium from nonpregnant (NP) and pregnant women. A typical blot is shown on the top. *p<0.05 vs. NP. n = 5–7 samples in each group. C. Gestation-dependent increase in SmAV expression in rat myometrium. A typical blot is shown on the top. *p<0.05 compared to NP, **p<0.01 compared to NP. ++ p<0.01 compared to D16. n = 4 in each group. NP, nonpregnant, D16 and D20, pregnant day 16 or day 20.</p

Smooth muscle Archvillin (SmAV) in pregnant human myometrium is localized with vinculin at cell surface.

<p>Term, not-in-labor, human uterine smooth muscle strips were microdissected and stretched to 2 fold slack length for 7 minutes. The fresh frozen sections (10 sections for each sample) were stained with rabbit anti-SmAV and mouse anti-vinculin. Alexa-dye-conjugated goat secondary antibodies were used. Left column, SmAV (red); middle column vinculin (green); right column, merged image. Top panels, scale bar, 20 µm. Lower 3 panels, expanded magnification of boxed area in top merged image. SmAV localizes with the dense plaque maker vinculin (inset, white arrows) at cell surface. Scale bar, 10 µm, n = 3.</p

Stretch of human myometrium directly increases <i>h-</i>CaD and ERK phosphorylation.

<p>A. h-CaD phosphorylation increases in response to stretch. Phosphorylated p-<i>h</i>-CaD is normalized to <i>h-</i>CaD protein level. *p<0.05 and ** p = 0.01 compared to resting sample (by ANOVA). n = 4 samples in each group. B. Stretch increases ERK2 phosphorylation. p-ERK2 signals are normalized to the total ERK2 protein levels. *p<0.05 compared to resting samples (by ANOVA). n = 4–5 samples in each group.</p

SmAV co-immunoprecipitates with phosphotyrosine, ERK and FAK.

<p>A. Myometrial SmAV associates with tyrosine-phosphorylated proteins in a stretch-dependent manner. Stretch and unstretched term pregnant human uterine smooth muscle homogenates were immunoprecipitated (IP) with anti-phospho-tyrosine antibody and immunoblotted (IB) with SmAV antibody. **p<0.01 compared to unstretched control samples. The insert is a typical SmAV western blot with IP samples. n = 5. B. Myometrial SmAV associates with ERK protein in a stretch- dependent manner. Stretched and unstretched term samples were immunoprecipitated with anti- ERK antibody and immunoblotted with anti-SmAV antibody. SmAV densitometry was normalized to input signals. n = 6. **p<0.01, ***p<0.001. C. Confirmation of stretch induced ERK phosphorylation in human myometrium. Stretch and unstretched samples were immunoprecipitated with anti- ERK antibody and immunoblotted with phospho-ERK antibody. The p-ERK2 infrared signals were also normalized to input signals. n = 6. **p<0.01. D. Myometrial ERK associates with FAK in a stretch-dependent manner. Stretch and unstretched samples homogenates were immunoprecipitated with anti- ERK antibody and immunoblotted with phospho-FAK⁹²⁵ antibody. The p-FAK infrared signals were normalized to input signals. n = 6. ***p<0.001</p

Identification of 100 KD protein tyrosine-phosphorylation in response to myometrial stretch.

<p>A. Stretch-induced tyrosine phosphorylation in human myometrium. Term pregnant uterine smooth muscle strips unstretched or stretched to 2x slack length for 7 min. Coomassie blue staining of anti-phosphotyrosine immunoprecipitates separated by 10% SDS-polyacrylamide gel electrophoresis (n = 2). B. Mass spectrometric identification of the 100 kD band. The protein band was excised from the gel. There is a 281/911 (31%) sequence coverage with 23 peptides matched to alpha 4 actinin, (in yellow) and 19% sequence coverage with 13 peptides matched to alpha 1 actinin (not shown). C. Alpha actinin is tyrosine phosphorylated by stretch in human myometrium. Homogenates of stretched and unstretched term pregnant human uterine smooth muscle were immunoprecipitated (IP) with an anti-phospho-tyrosine antibody and western immunoblotted (IB) with an alpha-actinin antibody. The graph shows the average densitometry from 5 independent experiments. The inserts are typical western blots of immunoprecipitates. *p<0.05 compared to unstretched control samples.</p

Model of signaling pathways involved in myometrial ERK activation.

<p>In addition to classical G-protein coupled receptors (GPCR) pathway described by others, data presented here suggest that the activation of ERK by focal adhesion signaling also promotes myometrial contraction and contributes to the initiation of labor. p: phosphorylation.</p

Stretch-induced tyrosine phosphorylation in human myometrium.

<p>A. A typical anti-phosphotyrosine immunoblot. Term pregnant uterine smooth muscle strips unstretched or stretched to 2x slack length for 2 min. The tissue homogenates were immunoblotted with an anti-tyrosine phosphorylation antibody. B–D. Term pregnant uterine smooth muscle strips were unstretched (rest) or stretched to 2x slack length at indicated time period. Average densitometry of phospho-tyrosine bands of 130 kD, 125 kD and 100 kD. n = 3–5 samples in each group.</p

<i>In vitro</i> stretch increases contractility in pregnant myometrium.

<p>Uterine contractility was measured and quantified as the area under curve (AUC). AUC is the integral of the <i>active</i> force over a period time of 2 or 7 minutes before and after stretch. Dotted line indicates level of passive stretch. Data presented in the bar graph were collected from 8 term, not in labor, pregnant human myometrial samples and total 17 smooth muscle strips. ++ p<0.01 compared to the AUC of spontaneous contraction before 2 minute-stretch. ** p<0.01 compared to the AUC of spontaneous contraction before 7 minute-stretch. An insert was the representative myograph recording of contractile force from term pregnant human myometrium in response to <i>in vitro</i> stretch.</p

Gestation-dependent changes in CaD content, calcium sensitivity and CaD phosphorylation.

<p>A. <i>h-</i>CaD expression increases during pregnancy. n = 3–7 samples in each group. * p<0.05 compared to NP. NP: nonpregnant. A single representative blot is illustrated on the top. To minimize the variation of the signal intensity between different Western blotting experiments, a reference sample was used in each blot for normalization since multiple samples were used from different pregnant women. B. Myometrial calcium sensitivity decreases with gestation. Human nonpregnant and pregnant not in labor uterine strips were permeabilized with alpha-toxin. Contractile force at 10⁻⁷ M calcium is expressed as a percentage of contraction induced by 10⁻⁵ M calcium. n = 3–5 samples in each group ** p<0.001 compared to NP. C. Increased CaD phosphorylation at Ser⁷⁸⁹ occurs only with the onset of labor. Phosphorylated p-<i>h</i>-CaD is normalized to <i>h-</i>CaD protein level. A single representative blot is included on the top. NP: nonpregnant, NIL: term, not in labor, IL: term, in labor. The samples used in experiments for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007489#pone-0007489-g001" target="_blank">Fig 1A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007489#pone-0007489-g001" target="_blank">Fig 1C</a> are different. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007489#pone-0007489-g001" target="_blank">Figure 1C</a> is an independent experiment. *p<0.05 vs. NP. n = 5–7 samples in each group.</p

Identification of p125 as FAK and p130 as Cas.

<p>A. Stretch selectively activates FAK at tyrosine site 925, not site 397. Term pregnant uterine smooth muscle strips were unstretched or stretched to 2x slack length at indicated time period. The tissue homogenates were probed with FAK site-specific antibodies. Phospho-FAK signals are normalized to the total FAK protein or alpha-Tubulin. *p<0.05 compared to unstretched control samples (by ANOVA). n = 3–5 in each group. Typical blots were shown on the top. Both FAK total protein antibody and FAK-Y925 phospho antibody are rabbit polyclonal antibodies, thus, a-tubulin mouse monoclonal antibody was chosen for normalization of the FAK-Y925 signal. B. Stretch activates pCas130. Phospho-Cas signals are normalized to Cas protein levels and expressed as p-Cas/Cas ratios. *p<0.05 compared to unstretched control samples (by ANOVA). n = 3–5 samples in each group.</p