Data Mining and Validation of AMPK Pathway as a Novel Candidate Role Affecting Intramuscular Fat Content in Pigs

Intramuscular fat (IMF) is an important economic trait for pork quality and a complex quantitative trait regulated by multiple genes. The objective of this work was to investigate the novel transcriptional effects of a multigene pathway on IMF deposition in the longissimus dorsi (LD) muscles of pigs. Potential signaling pathways were screened by mining data from three gene expression profiles in the Gene Expression Omnibus (GEO) database. We designed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) arrays for the candidate signaling pathways to verify the results in the LD muscles of two pig breeds with different IMF contents (Large White and Min). Western blot analysis was used to detect the expression levels of several candidate proteins. Our results showed that the AMPK signaling pathway was screened via bioinformatics analysis. Ten key hub genes of this signaling pathway (AMPK, ADIPOR1, ADIPOR2, LKB1, CAMKKβ, CPT1A, CPT1B, PGC-1α, CD36, and ACC1) were differentially expressed between the Large White and Min pigs. Western blot analysis further confirmed that LKB1/CaMKK2-AMPK-ACC1-CPT1A axis dominates the activity of AMPK signaling pathway. Statistical analyses revealed that AMPK signaling pathway activity clearly varied among the two pig breeds. Based on these results, we concluded that the activation of the AMPK signaling pathway plays a positive role in reducing IMF deposition in pigs.

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The AMPK signaling pathway is associated with the intramuscular fat trait in pigs

10.7287/peerj.preprints.26976v1 ◽

2018 ◽

Author(s):

Chaogang Yao ◽

Daxin Pang ◽

Chao Lu ◽

Aishi Xu ◽

Peixuan Huang ◽

...

Keyword(s):

Signaling Pathways ◽

Signaling Pathway ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Bioinformatic Analysis ◽

Intramuscular Fat ◽

Large White ◽

Positive Role ◽

Ampk Signaling ◽

Pig Breeds

Background. Intramuscular fat (IMF) is an important economic trait for pork quality and a complex quantitative trait regulated by multiple genes. The objective of this work was to investigate the novel transcriptional effects of a multigene pathways on IMF deposition in the longissimus dorsi(LD) muscles of pigs. Methods. Potential signaling pathways were screened by mining data from three gene expression profiles in the GEO database. We designed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) arrays for the candidate signaling pathways to verify the results in the LD muscles of three pig breeds with different IMF contents(Large White, Berkshire and Min). Results. The AMPK signaling pathway was screened via bioinformatic analysis. Ten key hub genes of this signaling pathway(AMPK, ADIPOR1, ADIPOR2, LKB1, CAMKKβ, CPT1A, CPT1B, PGC-1α, CD36 and ACC1) were differentially expressed. Statistical analyses revealed that AMPK pathway activity clearly varied among the three pig breeds. Conclusion. Based on these results, we concluded that the activation of the AMPK signaling pathway plays a positive role in reducing IMF deposition in pigs.

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The AMPK signaling pathway is associated with the intramuscular fat trait in pigs

10.7287/peerj.preprints.26976 ◽

2018 ◽

Author(s):

Chaogang Yao ◽

Daxin Pang ◽

Chao Lu ◽

Aishi Xu ◽

Peixuan Huang ◽

...

Keyword(s):

Signaling Pathways ◽

Signaling Pathway ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Bioinformatic Analysis ◽

Intramuscular Fat ◽

Large White ◽

Positive Role ◽

Ampk Signaling ◽

Pig Breeds

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Abstract 281: Resveratrol Enhances Cholesterol Efflux Through PPAR-γ--Dependent Signaling Pathways in Human Macrophages

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.32.suppl_1.a281 ◽

2012 ◽

Vol 32 (suppl_1) ◽

Author(s):

Ofek Y Hai ◽

Iryna Voloshyna ◽

Michael J Littlefield ◽

Steven Carsons ◽

Allison B Reiss

Keyword(s):

Gene Expression ◽

Western Blot ◽

Gene Silencing ◽

Signaling Pathways ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cholesterol Efflux ◽

Foam Cell Formation ◽

Mrna Levels ◽

Ppar Γ

Introduction: We previously reported that resveratrol, a plant derived polyphenol with numerous cardioprotective properties, improves cholesterol homeostasis by upregulating the expression of reverse cholesterol transport (RCT) proteins critical for preventing lipid overload and macrophage foam cell formation. However, the mechanism(s) by which resveratrol exerts this effect are unclear. The present study explores a possible mechanism for the atheroprotective actions of resveratrol on cholesterol metabolism. Hypothesis: We hypothesize that the anti-atherogenic effects of resveratrol are mediated through PPAR-γ dependent signaling pathways leading to upregulation of RCT proteins and enhanced cholesterol efflux. Methods: THP-1 macrophages, a pertinent model for atherosclerosis, were incubated for 20h with resveratrol (10μM) with/without the specific PPAR-γ antagonist GW6992 (1μM). Alternatively, cells were transfected with 100 nM of human PPAR-γ small interfering RNA (siRNA) for gene silencing prior to resveratrol treatment. Total RNA was isolated, converted to cDNA, and evaluated by QRT-PCR. Each reaction was done in triplicate. Western blot analysis was performed to confirm results of gene expression. Results: Resveratrol significantly increased expression of PPAR-γ, ABCA1, and 27-hydroxylase mRNA (mean±SEM, 136.2±8.5%, 168.3±9.2%, and 171.7±11.0% of control respectively, P<0.001). PPAR-γ gene silencing and PPAR-γ antagonist GW6992 effectively reduced PPAR-γ message by 90.4% and 54.7%, respectively (P<0.001). Both pharmacological blockade and gene knockdown of PPAR-γ nullified resveratrol effects on cholesterol efflux proteins. In cells treated with GW6992, mRNA levels of ABCA1 and 27-hydroxylase were decreased to 66.1±3.3% and 55.0±2.9% of control, respectively (P<0.001). Similarly, PPAR-γ silencing resulted in downregulation of ABCA1 and 27-hydroxylase expression to the level of control, which is significantly lower than in resveratrol treated cells (P<0.001). Data from gene expression studies were subsequently confirmed by Western blot analysis. Conclusions: We propose here a mechanistic model for the atheroprotective effects of resveratrol. Our data strongly suggests that resveratrol regulates cholesterol efflux and intracellular cholesterol processing via the PPAR-γ/LXR-α pathway.

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Zeaxanthin Exhibits Protective Effects in Myocardial Injury by Inhibiting TGF-β/Smad2/3 and p38MAPK/NF-κB Signaling Pathways

10.21203/rs.3.rs-966160/v1 ◽

2021 ◽

Author(s):

Mingyang Li ◽

Xiang Song ◽

Lichun Qi ◽

Yanhui Gao ◽

Xin Wang ◽

...

Keyword(s):

Western Blot ◽

Signaling Pathways ◽

Signaling Pathway ◽

Blot Analysis ◽

Western Blot Analysis ◽

Myocardial Injury ◽

Transforming Growth Factor ◽

Cardiomyocyte Apoptosis ◽

Related Protein ◽

Tgf Β1

Abstract Background: Zeaxanthin is a newly discovered natural product in β-carotenoid family with multiple bioactivities. Recently, it has been shown that zeaxanthin may have cardioprotective effects in several studies, but its mechanisms have not been fully investigated. Herein, we explored the role and mechanism of zeaxanthin in myocardial injury.Methods and Results: In this study, three different models were used to investigate the mechanism by which zeaxanthin alleviates myocardial injury. H9C2 Cardiomyocyte injury models were induced by H2O2. TUNEL assay, Flow cytometry, and Western blot analysis showed that treatment with zeaxanthin significantly decreased cardiomyocyte apoptosis and apoptosis-related protein expression. And reactive oxygen species (ROS) measurement analysis and Western blot analysis showed that treatment with zeaxanthin also could reduce the production of ROS and affect the expression of p38-Mitogen activated protein kinase/nuclear factor-κ gene bindin (p38MAPK/NF-κB) signaling pathway. Transforming Growth Factor-β1 (TGF-β1) was used to establish the fibrosis model in cardiac fibroblasts (CFs). QRT-PCR and Western blot analysis showed that treatment with zeaxanthin significantly decreased the expression of fibrosis markers in CFs. Myocardial injury animal models were induced by high-fat diet (HFD). Our results demonstrated that zeaxanthin improved fibrosis damage and cardiomyocyte apoptosis in HFD mice. Furthermore, Western blot analysis showed that TGF-β/Drosophila mothers against decapentaplegic2/3 (TGF-β/Smad2/3) signaling pathway related protein p-Smad2/3, Smad2/3, and TGF-β1 were significantly downregulated by zeaxanthin treatment.Conclusions: Zeaxanthin may alleviate HFD and H2O2-induced heart injury by regulating TGF-β/Smad2/3 and p38MAPK/NF-κB signaling pathways, which is of immense clinical significance in the treatment of cardiovascular disease.

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Punicalagin Prevents Inflammation in LPS-Induced RAW264.7 Macrophages by Inhibiting FoxO3a/Autophagy Signaling Pathway

Nutrients ◽

10.3390/nu11112794 ◽

2019 ◽

Vol 11 (11) ◽

pp. 2794 ◽

Cited By ~ 8

Author(s):

Cao ◽

Chen ◽

Ren ◽

Zhang ◽

Tan ◽

...

Keyword(s):

Western Blot ◽

Signaling Pathway ◽

Inflammatory Cytokines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Inflammatory Responses ◽

Raw264.7 Macrophages ◽

Tnf Α ◽

Autophagy Inhibition ◽

Pro Inflammatory Cytokines

Punicalagin, a hydrolysable tannin of pomegranate juice, exhibits multiple biological effects, including inhibiting production of pro-inflammatory cytokines in macrophages. Autophagy, an intracellular self-digestion process, has been recently shown to regulate inflammatory responses. In this study, we investigated the anti-inflammatory potential of punicalagin in lipopolysaccharide (LPS) induced RAW264.7 macrophages and uncovered the underlying mechanisms. Punicalagin significantly attenuated, in a concentration-dependent manner, LPS-induced release of NO and decreased pro-inflammatory cytokines TNF-α and IL-6 release at the highest concentration. We found that punicalagin inhibited NF-κB and MAPK activation in LPS-induced RAW264.7 macrophages. Western blot analysis revealed that punicalagin pre-treatment enhanced LC3II, p62 expression, and decreased Beclin1 expression in LPS-induced macrophages. MDC assays were used to determine the autophagic process and the results worked in concert with Western blot analysis. In addition, our observations indicated that LPS-induced releases of NO, TNF-α, and IL-6 were attenuated by treatment with autophagy inhibitor chloroquine, suggesting that autophagy inhibition participated in anti-inflammatory effect. We also found that punicalagin downregulated FoxO3a expression, resulting in autophagy inhibition. Overall these results suggested that punicalagin played an important role in the attenuation of LPS-induced inflammatory responses in RAW264.7 macrophages and that the mechanisms involved downregulation of the FoxO3a/autophagy signaling pathway.

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Fucoxanthin Inhibits Myofibroblast Differentiation and Extracellular Matrix Production in Nasal Polyp-Derived Fibroblasts via Modulation of Smad-Dependent and Smad-Independent Signaling Pathways

Marine Drugs ◽

10.3390/md16090323 ◽

2018 ◽

Vol 16 (9) ◽

pp. 323 ◽

Cited By ~ 3

Author(s):

Hyun Jung ◽

Dae-Sung Lee ◽

Seong Park ◽

Jung Choi ◽

Won-Kyo Jung ◽

...

Keyword(s):

Western Blot ◽

Signaling Pathways ◽

Nasal Polyp ◽

Blot Analysis ◽

Western Blot Analysis ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Type I ◽

Molecular Signaling ◽

Tgf Β1

Nasal polyps (NPs) are a multifactorial disorder associated with a chronic inflammatory state of the nasal mucosa. Fucoxanthin (Fx) is a characteristic orange carotenoid obtained from brown algae and has diverse immunological properties. The present study investigated whether Fx inhibits fibrosis-related effects in nasal polyp-derived fibroblasts (NPDFs) and elucidated the molecular signaling pathways involved. The production of collagen type I (Col-1) was investigated in NP tissue via immunohistochemistry and western blot analysis. NPDFs were treated with transforming growth factor (TGF)-β1 (1 ng/mL) in the presence or absence of Fx (5–30 µM). The levels of α-smooth muscle actin (α-SMA), Col-1, and phosphorylated (p)-Smad 2/3, signal protein-1 (SP-1), MAPKs (mitogen-activated protein kinases), and Akt were measured by western blot analysis. The expression of Col-1 was detected in NP tissues. TGF-β1 stimulated the production of α-SMA and Col-1, and stimulated the contraction of collagen gel. However, pretreatment with Fx attenuated these effects. Furthermore, these inhibitory effects were mediated through modulation of both Smad 2/3 and Akt/SP-1 signaling pathways in TGF-β1-induced NPDFs. The results from the present study suggest that Fx may be a novel anti-fibrotic agent for the treatment of NP formation.

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Monoclonal Antibody Against ROR1 in Chronic Lymphocytic Leukemia Cells Induced Apoptosis Via PI3-Kinase/AKT/CREB Pathway

Blood ◽

10.1182/blood.v120.21.1769.1769 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1769-1769

Author(s):

Amir Hossein Daneshmanesh ◽

Mohammad Hojjat-Farsangi ◽

Asa Sandin ◽

Abdul Salam Khan ◽

Ali Moshfegh ◽

...

Keyword(s):

Flow Cytometry ◽

Western Blot ◽

Signaling Pathways ◽

Blot Analysis ◽

Western Blot Analysis ◽

Pi3 Kinase ◽

Signaling Proteins ◽

Downstream Signaling ◽

Induced Apoptosis ◽

Creb Pathway

Abstract Abstract 1769 Background: Phosphoinositide 3-kinase (PI3K)/AKT cascade regulates cell survival, proliferation and differentiation in a variety of cells. In CLL cells PI3K pathway is constitutively activated leading to AKT activation and phosphorylation of cAMP response element-binding protein (CREB). CREB is a transcription factor overexpressed and constitutively phosphorylated in a variety of cancers and seems to have a role in tumor pathobiology. There is a great need to develop novel strategies for targeted therapy in CLL. Monoclonal antibodies (mAbs) specifically targeting leukemic cells might be a rewarding approach. ROR1 is a type I transmembrane receptor tyrosine kinase belonging to one of the twenty families of receptor tyrosine kinases (RTKs). ROR1 is overexpressed on CLL cells but not in white blood cells of healthy donors. ROR1 is constitutively phosphorylated in CLL and siRNA transfection induced apoptosis. We have developed a unique anti-ROR1 mAb directed against CRD (cysteine-rich domain) of the extracellular region of ROR1 capable of inducing direct apoptosis of primary CLL cells. Our anti-CRD mAb induced dephosphorylation of the ROR1 molecule. Aims: To study the apoptotic effect of an anti-ROR1 CRD mAb and effects on downstream signaling pathways involved in CLL, specially the PI3-kinase/AKT/CREB pathway using primary CLL cells. Methods: Using a peptide-based mouse mAb generation method we produced several mAbs against the three extracellular domains of ROR1. In the current study we used one of the best anti-ROR1 antibodies, an anti-CRD mAb raised against the CRD region of ROR1 (Daneshmanesh et al., Leukemia. 2012 Jun;26(6):1348-55). Flow cytometry was used for surface staining of ROR1. Primary CLL cells were incubated with the anti-ROR1 CRD mAb and apoptosis was detected by the MTT assay and Annexin V/propidium iodide (flow cytometry) methods in a 24 h assay. Antibody untreated and treated cell lysates were prepared and subjected to Western blot analysis for identification of signaling molecules involved in apoptosis induced by the anti-ROR1 CRD mAb. We analysed total and phosphorylated levels of the following signaling proteins: AKT, p-AKT, PI3K, p-PI3K, CREB, p-CREB, ERK, p-ERK, PKC and p-PKC. Phosphoproteins were measured before incubation with the mAb and after 20 min-2 h. Results: ROR1 surface expression was detected on 80–85% of the CLL cells. The frequency of apoptotic cells induced by the anti-CRD mAb was in the range of 45–50% which is in accordance with our previous reports (see above). Time kinetics experiments using anti-ROR1 CRD mAb incubated with primary CLL cells revealed dephosphorylation of ROR1 downstream signaling molecules. We analysed the following molecules known to be involved in CLL: PKC, PI3-kinase and ERK1/2. After co-culturing CLL cells with the anti-ROR1 CRD mAb, Western blot analysis showed decreased level of phosphorylated AKT in treated compared to untreated samples. No changes in the phosphorylation levels of ERK1/2 and PKC proteins were seen. Furthermore, we analysed the PI3-kinase protein which is upstream of AKT, and noticed that in CLL cells treated with the anti-ROR1 CRD mAb, the phosphorylation intensity of PI3-kinase p85 isoform has decreased but not p55 isoforrn. Moreover, we also studied the CREB phosphorylation in treated and untreated CLL samples and detected dephosphorylation of CREB in treated as compared to untreated samples. Conclusion: Incubation of CLL cells with an anti-ROR1 CRD mAb induced apoptosis of primary CLL cells. Apoptosis was preceded by dephosphorylation within 2 h of PI3-kinase, AKT and CREB proteins indicating deactivation of these signaling proteins by the anti-ROR1 mab. In untreated CLL cells no effect on phosphorylation of these proteins was noted. Furthermore our ROR1 mAb did not dephosphorylate PKC or ERK. Our data may suggest that activation of CREB molecule might occur via the PI3K/AKT pathway and may be a survival signal in CLL cells associated with the aberrant expression of ROR1. The constitutive phosphorylation of PKC and ERK1/2 seen in CLL might not be related to the overexpression of ROR1. Further studies are warranted for a better understanding of signaling pathways associated with ROR1 and the downstream signaling effects of ROR1 targeting drugs. Disclosures: No relevant conflicts of interest to declare.

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Endoplasmic Reticulum Stress is Involved in DFMO Attenuating Isoproterenol-Induced Cardiac Hypertrophy in Rats

Cellular Physiology and Biochemistry ◽

10.1159/000443096 ◽

2016 ◽

Vol 38 (4) ◽

pp. 1553-1562 ◽

Cited By ~ 5

Author(s):

Yan Lin ◽

Xiaojie Zhang ◽

Wei Xiao ◽

Bo Li ◽

Jun Wang ◽

...

Keyword(s):

Gene Expression ◽

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Cardiac Hypertrophy ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Cardiac Fibrosis ◽

Regulation Of Gene Expression ◽

Tunel Staining

Background/Aims: Studies performed in experimental animals have shown that polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with regulation of gene expression. Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, has attracted considerable interest for its antiproliferative role, which it exerts through inhibition of the polyamine pathway and cell turnover. Whether DFMO attenuates cardiac hypertrophy through endoplasmic reticulum stress (ERS) is unclear. Methods: Myocardial hypertrophy was simulated by isoproterenol (ISO). Polyamine depletion was achieved using DFMO. Hypertrophy was estimated using the heart/body index and atrial natriuretic peptide (ANP) gene expression. Cardiac fibrosis and apoptosis were measured by Masson and TUNEL staining. Expression of ODC and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blot analysis. Protein expression of ERS and apoptosis factors were analyzed using Western blot analysis. Results: DFMO treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO down-regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up-regulated the expression of SSAT and Bcl-2. Finally, these changes were partially reversed by the addition of exogenous putrescine. Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

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Secretoneurin is a potential paracrine factor from lactotrophs stimulating gonadotropin release in the goldfish pituitary

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00407.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. R1290-R1297 ◽

Cited By ~ 11

Author(s):

E. Zhao ◽

Caleb L. Grey ◽

Dapeng Zhang ◽

Jan A. Mennigen ◽

Ajoy Basak ◽

...

Keyword(s):

Gene Expression ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Time Course ◽

Pcr Analysis ◽

Pituitary Cells ◽

Mrna Levels ◽

Paracrine Factor ◽

Lh Release

Secretoneurin (SN) is a functional neuropeptide derived from the evolutionarily conserved part of precursor protein secretogranin II (SgII). In the time course study, SN (10 nM) stimulates luteinizing hormone (LH) production and secretion after 6 h of static incubation of goldfish pituitary cells. Due to the existence of SN-immunoreactivity (SN-IR) in goldfish lactotrophs, endogenous SN might exert a paracrine effect on LH in the pituitary. In an in vitro immunoneutralization experiment, coincubation with anti-SN antiserum reduces the stimulatory effect of salmon gonadotropin-releasing hormone (sGnRH) on LH release by 64%. Using Western blot analysis, we demonstrate that sGnRH significantly increases the expression of the major SgII-derived peptide (∼57 kDa, with SN-IR) and prolactin (PRL) after 12 h in the static culture of goldfish pituitary cells. Furthermore, there exists a significant correlation between the levels of these two proteins ( R = 0.76, P = 0.004). Another ∼30 kDa SgII-derived peptide containing SN is only observed in sGnRH-treated pituitary cells. Consistent with the Western blot analysis results, real-time RT-PCR analysis shows that a 12-h treatment with sGnRH induced 1.6- and 1.7-fold increments in SgII and PRL mRNA levels, respectively. SgII gene expression was also associated with PRL gene expression ( R = 0.66; P = 0.02). PRL cells loaded with the calcium-sensitive dye, fura 2/AM, respond to sGnRH treatment with increases in intracellular Ca2+ concentration level, suggesting a potential mechanism of GnRH on PRL cells and thus SgII processing and SN secretion. Taken together, endogenous lactotroph-generated SN, under the control of hypothalamic GnRH, exerts a paracrine action on neighboring gonadotrophs to stimulate LH release.

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Myc-epitope tagged proteins detected with the 9E10 antibody in immunofluorescence and immunoprecipitation assays but not in Western blot analysis

Biochemistry and Cell Biology ◽

10.1139/o98-003 ◽

1998 ◽

Vol 76 (1) ◽

pp. 125-128 ◽

Cited By ~ 10

Author(s):

Huizhou Fan ◽

Cristy Villegas ◽

Arthur K Chan ◽

Jim A Wright

Keyword(s):

Gene Expression ◽

Monoclonal Antibody ◽

Recombinant Protein ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Immunofluorescence Assay ◽

Expression Vectors ◽

Epitope Recognition ◽

In Cells

A human Myc epitope is frequently used to tag proteins for expression experiments in nonhuman cells. We used the monoclonal 9E10 antibody specific for this epitope to analyse the expression of four proteins carrying the Myc tag in cells transfected with expression vectors. While all four proteins can be detected by immunofluorescence and immunoprecipitation assays, surprisingly, only two proteins could be detected in Western blot analysis, indicating that epitope recognition by the monoclonal antibody can be blocked in some membrane-retained ectopic proteins. Other techniques such as immunofluorescence and immunoprecipitation assays can be successfully used with the 9E10 antibody to determine potential expression of Myc-tagged proteins.Key words: recombinant protein, Myc epitope, 9E10, Western blot, gene expression, immunofluorescence assay, immunoprecipitation.

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