Es were preabsorbed in 5 human serum in TBSTB at RT during the blocking process. Membranes were incubated for 1 h at RT with primary antibody solution. The HSP 70 (rabbit polyclonal antibody) was obtained from Enzo Life Sciences (ABI-SPA-812, lot: 09061120) and used at concentration of 1:1000. Membranes were washed and then incubated for 1 h at RT with horseradish peroxidase conjugated donkey anti-rabbit secondary antibody (Abcam (ab7083, lot: gr35152-1) diluted 1:3000 in TBSTB. Membranes were rinsed with TBSTB (265 min) and once with distilled water. Filters were 11967625 re-probed with a b-actin antibody (Sigma) to ensure even protein loading. Immunologically reactive proteins were visualised and quantified as described previously [13]. Statistical analysis was performed using MiniTab on a PC using analysis of variance (Kruskal Wallis for non-parametric data and ANOVA for normally distributed data). Comparison of groups was performed by the Mann Whitney test or student’s t-test as appropriate.Quantitative RT-PCRTotal RNA was isolated using the RNeasyH Midi Kit (Qiagen, 75142). RNA (100 ng) was reverse transcribed into cDNA. Buffers and primers were obtained from the QuantiTectH Kit (Qiagen, 205310) and GoScriptTM reverse transcriptase from Promega (A501C). HSP 70 (ID:NCBI 3303) expression (was analyzed by RT-PCR using validated TaqManH Gene Expression assays with StepOnePlus (Applied Biosystems). b-actin was used as an endogenous control. A positive control human placenta cDNA (Primer MedChemExpress PLV-2 design) was used. The relative target gene levels were calculated by comparative CT (DDCT). Statistical analysis was performed as above.Figure 2. Shows a representative Western blot analysis of HSP 70 expression in placenta of a patient (non-labor) and a patient in labor (n = 6 patients in each group for entire study). Samples are grouped according to distance sampled from cord insertion point. Four samples were obtained within each zone (see Figure 1). Molecular weight markers (kDa) are indicated by arrows. Also shown is a representative b-actin loading control for the gel above showing equal protein loading. doi:10.1371/journal.pone.0054540.gResultsTable 1 shows the demographics of the patients.Western BlottingThe first set of experiments was designed to test whether there was a difference in HSP 70 expression within individual placentae in both labor or non-labor. Figure 2 shows representative blots of HSP 70 expression in the area sampled 0? cm, 2? cm and 4?6 cm from the cord insertion point. The upper panel shows a placenta obtained from non-laboring caesarean section delivery. The bottom panel shows a placenta obtained from a women who was in labor and delivered vaginally. Figure 3 shows the results for the mean optical densities for HSP 70 expression for this set of experiments (6 patients in each group). The upper panel shows non-labor and the lower panel shows labor. Overall there was a significant difference between the 3 areas of the placenta for the non-labor group (ANOVA p = 0.008). There was Calciferol price significantly more HSP 70 expression in the 2? cm (middle) compared with the 4? cm (outer) area (student’s t-test, p = 0.03). No other differences were found (0? v 2?, p = 0.14) and (0? v 4?, p = 0.06). Overall there was also a significant difference between the 3 areas of the placenta in the labor group (ANOVA p = 0.002). There was significantly more HSP 70 expression in both the 0?2 cm and 2? cm areas compared with the 4? cm areas (p = 0.01 and p = 0.02 respe.Es were preabsorbed in 5 human serum in TBSTB at RT during the blocking process. Membranes were incubated for 1 h at RT with primary antibody solution. The HSP 70 (rabbit polyclonal antibody) was obtained from Enzo Life Sciences (ABI-SPA-812, lot: 09061120) and used at concentration of 1:1000. Membranes were washed and then incubated for 1 h at RT with horseradish peroxidase conjugated donkey anti-rabbit secondary antibody (Abcam (ab7083, lot: gr35152-1) diluted 1:3000 in TBSTB. Membranes were rinsed with TBSTB (265 min) and once with distilled water. Filters were 11967625 re-probed with a b-actin antibody (Sigma) to ensure even protein loading. Immunologically reactive proteins were visualised and quantified as described previously [13]. Statistical analysis was performed using MiniTab on a PC using analysis of variance (Kruskal Wallis for non-parametric data and ANOVA for normally distributed data). Comparison of groups was performed by the Mann Whitney test or student’s t-test as appropriate.Quantitative RT-PCRTotal RNA was isolated using the RNeasyH Midi Kit (Qiagen, 75142). RNA (100 ng) was reverse transcribed into cDNA. Buffers and primers were obtained from the QuantiTectH Kit (Qiagen, 205310) and GoScriptTM reverse transcriptase from Promega (A501C). HSP 70 (ID:NCBI 3303) expression (was analyzed by RT-PCR using validated TaqManH Gene Expression assays with StepOnePlus (Applied Biosystems). b-actin was used as an endogenous control. A positive control human placenta cDNA (Primer design) was used. The relative target gene levels were calculated by comparative CT (DDCT). Statistical analysis was performed as above.Figure 2. Shows a representative Western blot analysis of HSP 70 expression in placenta of a patient (non-labor) and a patient in labor (n = 6 patients in each group for entire study). Samples are grouped according to distance sampled from cord insertion point. Four samples were obtained within each zone (see Figure 1). Molecular weight markers (kDa) are indicated by arrows. Also shown is a representative b-actin loading control for the gel above showing equal protein loading. doi:10.1371/journal.pone.0054540.gResultsTable 1 shows the demographics of the patients.Western BlottingThe first set of experiments was designed to test whether there was a difference in HSP 70 expression within individual placentae in both labor or non-labor. Figure 2 shows representative blots of HSP 70 expression in the area sampled 0? cm, 2? cm and 4?6 cm from the cord insertion point. The upper panel shows a placenta obtained from non-laboring caesarean section delivery. The bottom panel shows a placenta obtained from a women who was in labor and delivered vaginally. Figure 3 shows the results for the mean optical densities for HSP 70 expression for this set of experiments (6 patients in each group). The upper panel shows non-labor and the lower panel shows labor. Overall there was a significant difference between the 3 areas of the placenta for the non-labor group (ANOVA p = 0.008). There was significantly more HSP 70 expression in the 2? cm (middle) compared with the 4? cm (outer) area (student’s t-test, p = 0.03). No other differences were found (0? v 2?, p = 0.14) and (0? v 4?, p = 0.06). Overall there was also a significant difference between the 3 areas of the placenta in the labor group (ANOVA p = 0.002). There was significantly more HSP 70 expression in both the 0?2 cm and 2? cm areas compared with the 4? cm areas (p = 0.01 and p = 0.02 respe.
