scholarly journals Determination of protein levels of browning genes in murine adipose tissues by western blotting. v1 (protocols.io.kbfcsjn)

protocols.io ◽  
2017 ◽  
Author(s):  
Bianca Hemmeryckx ◽  
Dries Bauters ◽  
H Roger
Keyword(s):  
Western Blotting ◽  
Adipose Tissues ◽  
Protein Levels

scholarly journals Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Ke Zhang ◽  
Zhuoying Li ◽  
Yunyang Lu ◽  
Linyi Xiang ◽  
Jiadong Sun ◽  
...  
Keyword(s):  
Western Blotting ◽  
Planar Cell Polarity ◽  
Type Ii Collagen ◽  
Mapk Signaling ◽  
Dependent Manner ◽  
Inflammatory Microenvironment ◽  
Functional Roles ◽  
Protein Levels ◽  
Oa Chondrocytes

Abstract Background The Wnt planar cell polarity (PCP) pathway is implicated in osteoarthritis (OA) both in animals and in humans. Van Gogh-like 2 (Vangl2) is a key PCP protein that is required for the orientation and alignment of chondrocytes in the growth plate. However, its functional roles in OA still remain undefined. Here, we explored the effects of Vangl2 on OA chondrocyte in vitro and further elucidated the molecular mechanism of silencing Vangl2 in Wnt5a-overexpressing OA chondrocytes. Methods Chondrocytes were treated with IL-1β (10 ng/mL) to simulate the inflammatory microenvironment of OA. The expression levels of Vangl2, Wnt5a, MMPs, and related proinflammatory cytokines were measured by RT-qPCR. Small interfering RNA (siRNA) of Vangl2 and the plasmid targeting Wnt5a were constructed and transfected into ATDC5 cells. Then, the functional roles of silencing Vangl2 in the OA chondrocytes were investigated by Western blotting, RT-qPCR, and immunocytochemistry (ICC). Transfected OA chondrocytes were subjected to Western blotting to analyze the relationship between Vangl2 and related signaling pathways. Results IL-1β induced the production of Vangl2, Wnt5a, and MMPs in a time-dependent manner and the significantly increased expression of Vangl2. Vangl2 silencing effectively suppressed the expression of MMP3, MMP9, MMP13, and IL-6 at both gene and protein levels and upregulated the expression of type II collagen and aggrecan. Moreover, knockdown of Vangl2 inhibited the phosphorylation of MAPK signaling molecules (P38, ERK, and JNK) and P65 in Wnt5a-overexpressing OA chondrocytes. Conclusions For the first time, we demonstrate that Vangl2 is involved in the OA process. Vangl2 silencing can notably alleviate OA progression in vitro by inhibiting the expression of MMPs and increasing the formation of the cartilage matrix and can inhibit the proinflammatory effects of Wnt5a via MAPK and NF-κB pathway. This study provides new insight into the mechanism of cartilage inflammation.

Simultaneous Determination of α-Fetoprotein and Immunoglobulin Levels From a Microhematocrit Capillary Tube

PEDIATRICS ◽  
1978 ◽  
Vol 61 (2) ◽  
pp. 301-303
Author(s):  
Alfried Kohlschütter
Keyword(s):  
Simultaneous Determination ◽  
Preterm Infants ◽  
Gestational Age ◽  
Capillary Tube ◽  
Long Island ◽  
Internal Diameter ◽  
Protein Levels ◽  
Immunoglobulin Levels ◽  
Blood Volumes

The α-fetoprotein (AFP) concentration in serum is an indicator of gestational age in preterm infants.1 IgM and IgA concentrations are helpful in the diagnosis of perinatal infections.2,3 Determination of all three protein levels can conveniently be made from one single microhematocrit capillary tube. The small blood volumes involved and the simplicity of the procedure make it a tool for routine use in newborn nurseries. The technique described can also be used for the determination of many other compounds. MATERIALS Heparinized microhematocrit capillary tubes (Propper Manufacturing Co., Inc., Long Island, N. Y.) are 75 mm long and have an internal diameter of 1.1 to 1.2 mm.

LncRNA Prostate Cancer Gene Expression Marker 1 (PCGEM1) Down-Regulation Inhibits the Development of Osteoarthritis by Modulating miR-152-3p

2022 ◽  
Vol 12 (2) ◽  
pp. 306-315
Author(s):  
Jie Song ◽  
Cheng Chen ◽  
Hui Zhang
Keyword(s):  
Cell Model ◽  
Luciferase Reporter ◽  
Down Regulation ◽  
Protein Levels ◽  
Diphenyltetrazolium Bromide ◽  
Proliferation And Apoptosis ◽  
Underlying Mechanisms ◽  
Commercial Kits ◽  
Related Proteins

Osteoarthritis (OA) is a chronic and inflammatory disease, leading to pain or even disability in severe cases. LncRNA PCGEM1 (PCGEM1) is reported to be dysregulated, serving as critical regulators in various human diseases, including OA. However, the biological role of PCGEM1 and its underlying mechanisms during OA remained unclear. In the present study, CHON-001 cells were exposed to interleukin (IL)-1β to construct the OA cell model. Expression of PCGEM1 and miR-152-3p in cells was determined by quantitative real-time polymerase chain reaction (qRT-PCR) assay. Corresponding commercial kits were used to measure the expressions of lactate dehydrogenase (LDH), inter-leukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. Protein levels of apoptosis-related proteins, cleaved-Caspase3 and Caspase3, were detected by Western blotting. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) tetrazolium (MTT) and flow cytometry assays were utilized for the determination of cell proliferation and apoptosis. The association between PCGEN1 and miR-152-3p was confirmed by a dual-luciferase reporter assay. From the results, PCGEM1 expression was significantly increased while miR-152-3p was inhibited in CHON-001 cells after IL-1β treatment. In addition, silencing of PCGEM1 could promote proliferation, inhibit the apoptosis, suppress LDH level and alleviate inflammation response caused by IL-1β in CHON-001 cells by sponging miR-152-3p. In a word, PCGEM1 down-regulation suppressed OA progression by the regulation of miR-152-3p expression, functioning as a potential therapeutic target for OA clinical treatment.

Abstract 3653: Akt3 Offers Stronger Protection than Akt1 Against Brain Injury by Differentially Promoting Mtor Protein Levels In Both In Vitro and In Vivo Stroke Models

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Rong Xie ◽  
Michelle Cheng ◽  
Mei Li ◽  
Robert Sapolsky ◽  
Heng Zhao
Keyword(s):  
Gene Transfer ◽  
Western Blotting ◽  
Ischemic Injury ◽  
Akt Pathway ◽  
Over Expression ◽  
Protein Levels ◽  
Akt Isoforms ◽  
The Brain

Background and Objective: Akt is a serine-threonine kinase that plays critical role in promoting cell survival. Akt consists of three isoforms (Akt1, 2, 3), with Akt3 predominantly expressed in the brain. Although Akt pathway has been shown to mediate neuronal survival in cerebral ischemic injury, it is unclear how these Akt isoforms contribute to neuronal protection, and whether exogenous Akt can protect the brain against ischemic injury or not. In this study, we over-expressed Akt isoforms and its downstream signaling proteins such as FKHR and PRAS40 to investigate the role of the Akt pathway along with its potential relationship with the mTOR pathway in stroke. Methods: Sprauge Dawley rats (250∼280g) were used for all studies. A lentiviral vector consists of a CMV promoter driving IRES-eGFP was used to clone an active Akt 1 and 3 (cAKt 1 and 3), dominant-negative Akt (AktDN), active FKHR (AAA FKHR), and PRAS40. Lentivirus expressing these genes were added to primary mixed cortical cultures for two days prior to oxygen glucose deprivation (OGD) (MOI=1:5). Neuronal survival was measured by LDH release. Lentivirus were stereotaxically injected into the cortex, and rats were subjected to focal ischemia induced by distal MCA occlusion combined with bilateral CCA occlusion. Western blotting and immunofluorescent confocal microscopy were used to detect the expression of Akt isoforms and other proteins in both the Akt and mTOR pathways. Results: Western blotting analysis showed that both endogenous Akt1 and 3 proteins degraded as early as 1 h after stroke, while Akt2 protein remained unchanged until 24 h after stroke. In vitro studies showed that over-expression of both constitutively active cAkt1 and cAkt3 decreased LDH release after OGD, while AktDN worsened neuronal death ( P <0.05). In vivo over-expression of cAkt1, cAkt3 and PRAS40 reduced infarct size after stroke ( P <0.01). Gene transfer of cAkt1 and 3 also promoted protein levels of pAkt (phosphorylated Akt), pPRAS40, pFKHR, pPTEN, pmTOR, but not pGSK3β. Both in vitro and in vivo studies showed that over-expression of cAkt3 resulted in a stronger protection than cAkt1 ( P <0.05). Interestingly, cAkt3 gene transfer preserved both endogenous protein levels of Akt1 and 3, whereas cAkt1 gene transfer only preserved endogenous Akt1. Furthermore, cAkt3 promoted higher pmTOR levels than cAkt1. Treatment of rapamycin, an mTOR inhibitor, blocked the protective effects of both cAkt1 and cAkt3 both in vitro and in vivo. Conclusion: Lentiviral-mediated overexpression of cAkt3 confers stronger protection than that of cAkt1, by maintaining both endogenous Akt1 and Akt3, as well as promoting higher mTOR activities after stroke.

Abstract 3126: Ischemic Postconditioning Facilitates Brain Recovery After Stroke by Promoting mTOR Activity

Stroke ◽  
2012 ◽  
Vol 43 (suppl_1) ◽  
Author(s):  
Peng Wang ◽  
Rong Xie ◽  
Xunming Ji ◽  
Heng Zhao
Keyword(s):  
Western Blotting ◽  
Brain Plasticity ◽  
Focal Ischemia ◽  
Mtor Pathway ◽  
Synaptic Function ◽  
Ischemic Postconditioning ◽  
Protein Levels ◽  
Left Lateral Ventricle ◽  
Brain Recovery ◽  
Mtor Activity

Introduction: Ischemic postconditioning represents a series of brief occlusions of blood flow before complete restoration of reperfusion after brain stroke. We have shown that ischemic postconditioning reduces infarct size in focal ischemia in rats and improves neurological deficits, but the underlying protective mechanisms are not fully understood. The mammalian target of rapamycin (mTOR) pathway plays a key role in cell growth, differentiation and survival. We studied the hypothesis that the mTOR pathway is involved in the protective effect of ischemic postconditioning and brain recovery after stroke in rats. Methods: Focal ischemia was induced by 30 min of bilateral CCA occlusion and permanent distal MCA occlusion in rats. Ischemic postconditioning was induced by 3 cycles of 30 sec reperfusion and 10 sec occlusion at the end of stroke. Rapamycin, an mTOR inhibitor, was injected into the left lateral ventricle 1 hour before stroke onset. For the behavior test, home cage and vibrissa-elicited limb use tests were used and performed until 21d after stroke. Peri-infarct tissues were collected 1 and 3 weeks after stroke for Western blotting and immunostaining. Molecular markers related with synaptic transmission and neuronal growth and plasticity, including Gap-43, synaptophysin, MAP-2 and PSD-95 proteins were detected. Protein levels of phosphorylated mTOR (p-MTOR) and 4EPB-1 in the mTOR pathway, and the upstream molecule, Akt, were also measured. Results: Ischemic postconditioning improved neurological function when measured 2 weeks after stroke (n=6, p<0.05), and reduced brain injury size by 34.2% (P<0.05). These protective effects were abolished by rapamycin treatment. The results of Western blotting showed that postconditioning substantially promoted the protein level of Gap-43, MAP-2 and PSD-95, but not synaptophysin. Rapamycin significantly inhibited Gap-43 levels at 1 and 3 weeks after stroke, and inhibited Map-2 level at 1 week (P<0.05). Postconditioning significantly increased the protein levels of p-Akt, p-mTOR, p-4EBP-1 compared with control ischemia (p<0.05) at 1 week after stroke injury. Rapamycin attenuated p-mTOR levels 1 and 3 weeks after stroke, and inhibited p-4EBP-1 level at 1 week (p<0.05), but had no effects on the expression level of p-Akt and Akt. Conclusion: Ischemic postconditioning improved brain function, which is consistent with the improved expression of proteins related with synaptic function and brain plasticity. This protection appears to be achieved by the enhanced mTOR activity.

scholarly journals Optimization of Chemoenzymatic Mass Tagging by Strain-Promoted Cycloaddition (SPAAC) for the Determination of O-GlcNAc Stoichiometry by Western Blotting

Biochemistry ◽  
2018 ◽  
Vol 57 (40) ◽  
pp. 5769-5774 ◽  
Author(s):  
Narek Darabedian ◽  
John W. Thompson ◽  
Kelly N. Chuh ◽  
Linda C. Hsieh-Wilson ◽  
Matthew R. Pratt
Keyword(s):  
Western Blotting

Abstract P207: Role of (Pro)Renin Receptor in the Pathogenesis of Colon Cancer

Hypertension ◽  
2015 ◽  
Vol 66 (suppl_1) ◽  
Author(s):  
Akira Nishiyama ◽  
Juan Wang ◽  
Shinichi Yachida ◽  
Genevieve Nguyen ◽  
Takuo Hirose ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Cell Number ◽  
Ductal Adenocarcinoma ◽  
Mrna Levels ◽  
Protein Levels ◽  
Colon Cancer Cell Lines ◽  
Cancer Tissues

(Pro)renin receptor ((P)RR) is a component of the Wnt receptor complex (Science, 2010). We have recently demonstrated that (P)RR plays an important role in the tumorigenesis of pancreatic ductal adenocarcinoma via the activation of Wnt/β-catenin signaling pathway (Shibayama et al. Sci Rep. 2015). Since the patients with colon cancer often show aberrantly activated Wnt/β-catenin-dependent signaling pathway by the mutations of its components, we investigated the possible role of (P)RR and Wnt/β-catenin signaling pathway in carcinogenesis of colon cancer. Real-time PCR was used for measuring mRNA levels of (P)RR. Protein levels of (P)RR was determined by Western blotting and immunohistochemistry. Activated β-catenin levels were determined by Western blotting. Cell proliferative ability was evaluated by counting the cell number in cultured colon cancer cell lines, HCT116 and DLD-1 cells. As compared to normal colon tissues (n=6), mRNA and protein levels of (P)RR were increased by 2.6- and 2.2-fold, respectively, in colon cancer tissues (n=9), which were associated with increased activated β-catenin levels (by 2.8-fold, P<0.05). However, plasma soluble (P)RR levels were not changed in patients with colon cancer (n=9). (P)RR and activated β-catenin levels were also increased in HCT116 (by 2.2- and 2.7-fold, n=5, respectively) and DLD-1 cells (by 1.9- and 2.8-fold, n=5, respectively). In these cells, inhibiting (P)RR with an siRNA attenuated the activity of β-catenin and reduced the proliferative abilities (n=5, P<0.05, respectively). These data suggest that (P)RR contributes to the tumorigenesis of colon cancer through the activation of Wnt/β-catenin signaling pathway.

Abstract WMP38: Inhibition of Rip1 Kinase Improves Brain Functional Recovery After Ischemic Stroke via Reducing Astrocytic Scar Formation

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Huiling Zhang ◽  
Zhong-Sheng Li ◽  
Yong Ni ◽  
Xian-Yong Zhou ◽  
Shi-Gang Qiao
Keyword(s):  
Ischemic Stroke ◽  
Western Blotting ◽  
Functional Recovery ◽  
Recovery Phase ◽  
Glial Scar ◽  
Scar Formation ◽  
Western Blotting Analysis ◽  
Protein Levels

During the recovery phase of ischemic stroke, one of the major barriers for the spontaneous neuronal axon regeneration is the formation of astrogliosis and glial scar, and targeting astrogliosis becomes a therapeutic strategy for ischemic stroke. However, the mechanism regulating the process of scar components after ischemia still remains poorly understood. The aim of this study was to observe the role of RIP1 kinase (RIP1K), the key regulator of necroptosis (programmed necrosis) in the brain functional recovery after ischemic stroke and in the ischemic stroke-induced astrogliosis and glial scar formation in both in vitro and in vivo glial scar models. The glial scar formation model in vitro or in vivo was established by using primary cultured astrocyte subjected to 6 hours of oxygen-glucose deprivation (OGD) following 12 hours or 24 hours reperfusion, or by 90 min of transient middle cerebral artery occlusion (tMCAO) and reperfusion in rats. Western blotting analysis and immunohistochemical assay showed that knockdown of RIP1K by lentivirally-delivered shRNAs against RIP1K (shRNA RIP1K) could decrease several protein levels of glial scar markers such as glial fibillary acidic protein (GFAP), neurocan and phosphacan both in in vitro and in vivo glial scar models. Furthermore, western blotting analysis showed that knockdown of RIP1K reduced the protein levels of VEGF-D receptor 3 in in vitro glial scar models. In addition, knockdown of RIP1K also notably reduced the shrinking volume and ameliorated the behavioral symptoms in the recovery phase of rats after tMCAO. And immunocytochemistry assay demonstrated that RIP1K knockdown promoted the neuronal axonal generation in a neuron and astrocyte co-culture system. Our data indicates that RIP1K might play an important role in the formation of glial scar after ischemic stroke via promoting the function of VEGF-D receptor 3 in astrocytes.

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