β-Actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients

Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

doi:10.1016/j.ab.2012.05.008

Cited by 28 publications

(18 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The blots were analyzed with Image Pro Plus. A plasma level of β-actin, which has been shown to be a least altered plasma protein (Zhang et al 2012), was used as loading control. The levels of Fg were assessed by measuring an integrated optical density (IOD) of its bands in each sample lane profile relative to the IOD of the respective β-actin band.…”

Section: Methodsmentioning

confidence: 99%

Ablation of matrix metalloproteinase-9 gene decreases cerebrovascular permeability and fibrinogen deposition post traumatic brain injury in mice

et al. 2014

View full text Add to dashboard Cite

Traumatic brain injury (TBI) is accompanied with enhanced matrix metalloproteinase-9 (MMP-9) activity and elevated levels of plasma fibrinogen (Fg), which is a known inflammatory agent. Activation of MMP-9 and increase in blood content of Fg (i.e. hyperfibrinogenemia, HFg) both contribute to cerebrovascular disorders leading to blood brain barrier disruption. It is well-known that activation of MMP-9 contributes to vascular permeability. It has been shown that at an elevated level (i.e. HFg) Fg disrupts blood brain barrier. However, mechanisms of their actions during TBI are not known. Mild TBI was induced in wild type (WT, C57BL/6J) and MMP-9 gene knockout (Mmp9−/−) homozygous, mice. Pial venular permeability to fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) in pericontusional area was observed 14 days after injury. Mice memory was tested with a novel object recognition test. Increased expression of Fg endothelial receptor intercellular adhesion protein-1 and formation of caveolae were associated with enhanced activity of MMP-9 causing an increase in pial venular permeability. As a result, an enhanced deposition of Fg and cellular prion protein (PrPC) were found in pericontusional area. These changes were attenuated in Mmp9−/− mice and were associated with lesser loss of short-term memory in these mice than in WT mice. Our data suggest that mild TBI-induced increased cerebrovascular permeability enhances deposition of Fg-PrPC and loss of memory, which is ameliorated in the absence of MMP-9 activity. Thus, targeting MMP-9 activity and blood level of Fg can be a possible therapeutic remedy to diminish vasculo-neuronal damage after TBI.

show abstract

Section: Methodsmentioning

confidence: 99%

Ablation of matrix metalloproteinase-9 gene decreases cerebrovascular permeability and fibrinogen deposition post traumatic brain injury in mice

et al. 2014

View full text Add to dashboard Cite

show abstract

“…housekeeping proteins, from the same samples. This procedure adjusts the intensities with respect to small variations in the number of cells and loading across samples within the same blot [11]–[13]. The need to normalise the data arises when comparing the results from biological replicates of the same experiment, for example to obtain statistical evidence that different conditions induce different protein amounts.…”

Section: Introductionmentioning

confidence: 99%

Evaluating Strategies to Normalise Biological Replicates of Western Blot Data

et al. 2014

View full text Add to dashboard Cite

Western blot data are widely used in quantitative applications such as statistical testing and mathematical modelling. To ensure accurate quantitation and comparability between experiments, Western blot replicates must be normalised, but it is unclear how the available methods affect statistical properties of the data. Here we evaluate three commonly used normalisation strategies: (i) by fixed normalisation point or control; (ii) by sum of all data points in a replicate; and (iii) by optimal alignment of the replicates. We consider how these different strategies affect the coefficient of variation (CV) and the results of hypothesis testing with the normalised data. Normalisation by fixed point tends to increase the mean CV of normalised data in a manner that naturally depends on the choice of the normalisation point. Thus, in the context of hypothesis testing, normalisation by fixed point reduces false positives and increases false negatives. Analysis of published experimental data shows that choosing normalisation points with low quantified intensities results in a high normalised data CV and should thus be avoided. Normalisation by sum or by optimal alignment redistributes the raw data uncertainty in a mean-dependent manner, reducing the CV of high intensity points and increasing the CV of low intensity points. This causes the effect of normalisations by sum or optimal alignment on hypothesis testing to depend on the mean of the data tested; for high intensity points, false positives are increased and false negatives are decreased, while for low intensity points, false positives are decreased and false negatives are increased. These results will aid users of Western blotting to choose a suitable normalisation strategy and also understand the implications of this normalisation for subsequent hypothesis testing.

show abstract

“…β-actin has relatively high and constant expression in the total protein of cancer cells. Therefore, β-actin has been widely employed in Western blotting as a loading control [ 17 ]. In this study, GAPDH and β-actin were also used as controls for RT-PCR and Western blot assays, respectively.…”

Section: Discussionmentioning

confidence: 99%

Anticancer Effect of Ursodeoxycholic Acid in Human Oral Squamous Carcinoma HSC-3 Cells through the Caspases

et al. 2015

View full text Add to dashboard Cite

Bear bile was used as a traditional medicine or tonic in East Asia, and ursodeoxycholic acid (UDCA) is the most important compound in bear bile. Further, synthetic UDCA is also used in modern medicine and nutrition; therefore, its further functional effects warrant research, in vitro methods could be used for the fundamental research of its anticancer effects. In this study, the apoptotic effects of UDCA in human oral squamous carcinoma HSC-3 cells through the activation of caspases were observed by the experimental methods of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, DAPI (4’,6-diamidino-2-phenylindole) staining, flow cytometry analysis, RT-PCR (reverse transcription-polymerase chain reaction) assay and Western blot assay after HSC-3 cells were treated by different concentrations of UDCA. With 0 to 400 μg/mL UDCA treatment, UDCA had strong growth inhibitory effects in HSC-3 cells, but had almost no effect in HOK normal oral cells. At concentrations of 100, 200 and 400 μg/mL, UDCA could induce apoptosis compared to untreated control HSC-3 cells. Treatment of 400 μg/mL UDCA could induce more apoptotic cancer cells than 100 and 200 μg/mL treatment; the sub-G1 DNA content of 400 μg/mL UDCA treated cancer cells was 41.3% versus 10.6% (100 μg/mL) and 22.4% (200 μg/mL). After different concentrations of UDCA treatment, the mRNA and protein expressions of caspase-3, caspase-8, caspase-9, Bax, Fas/FasL (Fas ligand), TRAIL (TNF-related apoptosis-inducing ligand), DR4 (death receptor 4) and DR5 (death receptor 5) were increased in HSC-3 cells, and mRNA and protein expressions of Bcl-2 (B-cell lymphoma 2), Bcl-xL (B-cell lymphoma-extra large), XIAP (X-linked inhibitor of apoptosis protein), cIAP-1 (cellular inhibitor of apoptosis 1), cIAP-2 (cellular inhibitor of apoptosis 2) and survival were decreased. Meanwhile, at the highest concentration of 400 μg/mL, caspase-3, caspase-8, caspase-9, Bax, Fas/FasL, TRAIL, DR4, DR5, and IκB-α expression levels were the highest, and Bcl-2, Bcl-xL, XIAP, cIAP-1, cIAP-2, survival, and NF-κB expression levels were the lowest. These results proved that UDCA could induce apoptosis of HSC-3 cancer cells through caspase activation, and the higher concentration of UDCA had stronger effects in vitro. UDCA might be a good nutrient for oral cancer prevention.

show abstract

β-Actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients

Cited by 28 publications

References 25 publications

Ablation of matrix metalloproteinase-9 gene decreases cerebrovascular permeability and fibrinogen deposition post traumatic brain injury in mice

Ablation of matrix metalloproteinase-9 gene decreases cerebrovascular permeability and fibrinogen deposition post traumatic brain injury in mice

Evaluating Strategies to Normalise Biological Replicates of Western Blot Data

Anticancer Effect of Ursodeoxycholic Acid in Human Oral Squamous Carcinoma HSC-3 Cells through the Caspases

Contact Info

Product

Resources

About