Stability of reference proteins in human placenta: General protein stains are the benchmark

Lanoix, Dave; St‐Pierre, Joey; Lacasse, Andrée-Anne; Viau, Mélanie; Lafond, Julie; Vaillancourt, Cathy

doi:10.1016/j.placenta.2011.12.008

Cited by 69 publications

(59 citation statements)

References 25 publications

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“…This is further complicated by the fact that some detection methods, in particular enhanced chemiluminescence using x-ray film, have a very restricted linear range, and careful attention to the experimental conditions is necessary to ensure linearity. It is typically better to normalize Western blots using total protein loading as the denominator (3)(4)(5)(6)(7)(8)(9)(10)(11). To avoid potential pitfalls and with a focus on improving transparency, the JBC strongly recommends that authors describe their methods used to quantify signal intensity, how the linearity of signal intensity with antigen loading was established, and how protein loading was normalized between lanes.…”

Section: Presentation and Quantitation Of Western Blotsmentioning

confidence: 99%

Transparency Is the Key to Quality

Fosang¹,

Colbran²

2015

Journal of Biological Chemistry

102

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Section: Presentation and Quantitation Of Western Blotsmentioning

confidence: 99%

Transparency Is the Key to Quality

Fosang¹,

Colbran²

2015

Journal of Biological Chemistry

102

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“…For protein loading control, blots were stained with 0.1% Ponceau-S in 5% acetic acid (Santa Cruz Biotechnology), rinsed in distilled water to clear background, then imaged using Quantity One software. 29 …”

Section: Western Blottingmentioning

confidence: 99%

TREM-1 Expression Is Increased in Human Placentas From Severe Early-Onset Preeclamptic Pregnancies Where It May Be Involved in Syncytialization

2014

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Preeclampsia, a major cause of maternal and perinatal morbidity and mortality, is thought to be attributable to dysregulation of trophoblast invasion and differentiation. Microarray studies have shown that triggering receptor expressed on myeloid cells (TREM) 1, a cell surface molecule involved in the inflammatory response, is increased in preeclamptic placentas. The aim of this study was to determine the level of TREM-1 expression in severe early-onset preeclamptic placentas and its functional role in trophoblast differentiation. Placenta was obtained from women with severe early-onset preeclampsia (n ¼ 19) and gestationally matched preterm controls placentas (n ¼ 8). The TREM-1 expression was determined by quantitative reverse transcriptase polymerase chain reaction and Western blotting. The effect of TREM-1 small interfering RNA on cell fusion and differentiation was assessed in BeWo cells. The effect of oxygen tension on TREM-1 levels, in basal or forskolin-treated BeWo cells, was also assessed. The TREM-1 was localized to the syncytiotrophoblast layer, and TREM-1 messenger RNA and protein expression was significantly increased in preeclamptic placentas. The BeWo cells treated with forskolin were associated with increased TREM-1 expression. The TREM-1 knockdown inhibited forskolin-induced expression of the differentiation marker b-human chorionic gonadotropin but had no effect on the cell-fusion marker E-cadherin. The increase in TREM-1 expression in BeWo cells treated with forskolin during normoxic conditions was reduced in forskolin-treated cells under hypoxic conditions. In conclusion, TREM-1 is increased in preeclamptic placentas and by forskolin treatment. Knockdown of TREM-1 by RNA interference inhibits cell differentiation but has no effect on cell-cell fusion. Finally, we show that TREM-1 upregulation is attenuated under hypoxic conditions in which cell differentiation is impaired.

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“…For SIRT6, two isoforms were present, and quantification was performed on both bands. Data were corrected for background and normalized to b-actin or Ponceau S staining (for nuclear protein [32]). …”

Section: Western Blot Analysismentioning

confidence: 99%

SIRT6 Is Decreased with Preterm Labor and Regulates Key Terminal Effector Pathways of Human Labor in Fetal Membranes1

Lim

Barker

Lappas

2013

Biology of Reproduction

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Preterm birth is a major determinant of neonatal mortality and morbidity, affecting approximately one-third of preterm births as a result of prelabor rupturing of membranes. Infection and inflammation have strong causal links to preterm delivery, resulting in the activation of nuclear factor-kappaB (NFKB) and its downstream targets. Human sirtuin (SIRT) 6, which has ADP-ribosyl transferase and deacetylase activity, exhibits anti-inflammatory actions. The aims of this study were to determine the effect of 1) human preterm labor on SIRT6 expression in human gestational tissue and 2) the effect in primary amnion cells of SIRT6 inhibition, using small interfering RNA (siRNA) on prolabor mediators. To determine the effect of human preterm labor on SIRT6 expression, human fetal membranes were collected from women at preterm at the time of Cesarean section (no labor; n = 9) and from women after spontaneous labor and delivery (n = 9). SIRT6 mRNA and protein expression were significantly lower in fetal membranes after spontaneous preterm labor. Transfection of primary amnion cells with SIRT6 siRNA was associated with an increase in IL-1beta-induced proinflammatory cytokine gene expression and release (IL6, IL8, TNF [TNF-alpha]), cyclooxygenase ([COX]-2; official symbol PTGS2) expression and subsequent prostaglandin (PGE(2) and PGF(2alpha)) release, and MMP9 gene expression and release of pro-MMP9. To determine whether SIRT6 affects NFKB transcriptional activity, primary amnion cells were transfected with NFKB tagged with luciferase and stimulated with IL1B. As expected, IL1B induced NFKB transcriptional activity. However, when cells were also cotransfected with a vector expressing SIRT6, there was a decrease in NFKB transcriptional activity. In conclusion, SIRT6 plays a role in regulating the terminal effector pathways of human labor and delivery via the NFKB pathway.

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Stability of reference proteins in human placenta: General protein stains are the benchmark

Cited by 69 publications

References 25 publications

Transparency Is the Key to Quality

Transparency Is the Key to Quality

TREM-1 Expression Is Increased in Human Placentas From Severe Early-Onset Preeclamptic Pregnancies Where It May Be Involved in Syncytialization

SIRT6 Is Decreased with Preterm Labor and Regulates Key Terminal Effector Pathways of Human Labor in Fetal Membranes1

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