Localization of the ANG II type 2 receptor  in the microcirculation of skeletal muscle

Only functional studies have suggested the presence of the ANG II type 2 (AT2) receptor in the microcirculation. To determine the distribution of this receptor in the rat skeletal muscle microcirculation, a polyclonal rabbit anti-rat antiserum was developed and used for immunohistochemistry and Western blot analysis. The antiserum was prepared against a highly specific and antigenic AT2-receptor synthetic peptide and was validated by competition and sensitivity assays. Western blot analysis demonstrated a prominent, single band at ∼40 kDa in cremaster and soleus muscle. Immunohistochemical analysis revealed a wide distribution of AT2 receptors throughout the skeletal muscle microcirculation in large and small microvessels. Microanatomic studies displayed an endothelial localization of the AT2 receptor, whereas dual labeling with smooth muscle α-actin also showed colocalization of the AT2 receptor with vascular smooth muscle cells. Other cells associated with the microvessels also stained positive for AT2 receptors. Briefly, this study confirms previous functional data and localizes the AT2 receptor to the microcirculation. These studies demonstrate that the AT2 receptor is present on a variety of vascular cell types and that it is situated in a fashion that would allow it to directly oppose ANG II type 1 receptor actions.

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Dystrophin in skeletal muscle I. Western blot analysis using a monoclonal antibody

Journal of the Neurological Sciences ◽

10.1016/0022-510x(89)90223-2 ◽

1989 ◽

Vol 94 (1-3) ◽

pp. 125-136 ◽

Cited By ~ 108

Author(s):

Louise V.B. Nicholson ◽

Keith Davison ◽

Gavin Falkous ◽

Colin Harwood ◽

Elizabeth O'Donnell ◽

...

Keyword(s):

Skeletal Muscle ◽

Monoclonal Antibody ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis

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The Branch Point Enzyme of the Mevalonate Pathway for Protein Prenylation Is Overexpressed in theob/ob Mouse and Induced by Adipogenesis

Molecular and Cellular Biology ◽

10.1128/mcb.20.6.2158-2166.2000 ◽

2000 ◽

Vol 20 (6) ◽

pp. 2158-2166 ◽

Cited By ~ 21

Author(s):

David Vicent ◽

Eleftheria Maratos-Flier ◽

C. Ronald Kahn

Keyword(s):

Skeletal Muscle ◽

Western Blot ◽

Branch Point ◽

Northern Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Northern Blot Analysis ◽

Geranylgeranyl Diphosphate ◽

Protein Prenylation ◽

Ggpp Synthase

ABSTRACT We have recently reported that skeletal muscle of theob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the expression of multiple genes. Analysis and cloning of a full-length cDNA of one of the overexpressed mRNAs revealed a 300-amino-acid protein that could be identified as the mouse geranylgeranyl diphosphate synthase (GGPP synthase) based on its homology to proteins cloned from yeast and fungus. GGPP synthase catalyzes the synthesis of all-trans-geranylgeranyl diphosphate (GGPP), an isoprenoid used for protein isoprenylation in animal cells, and is a branch point enzyme in the mevalonic acid pathway. Three mRNAs for GGPP synthase of 4.3, 3.2, and 1.7 kb were detected in Northern blot analysis. Western blot analysis of tissue homogenates using specific antipeptide antibodies revealed a single band of 34.8 kDa. Expression level of this protein in different tissues correlated with expression of the 4.3- and 3.2-kb mRNAs. GGPP synthase mRNA expression was increased 5- to 20-fold in skeletal muscle, liver, and fat of ob/obmice by Northern blot analysis. Western blot analysis also showed a twofold overexpression of the protein in muscle and fat but not in liver, where the dominant isoform is encoded by the 1.7-kb mRNA. Differentiation of 3T3-L1 fibroblasts into adipocytes induced GGPP synthase expression more than 20-fold. Using the immunoprecipitated protein, we found that mammalian GGPP synthase synthesizes not only GGPP but also its metabolic precursor farnesyl diphosphate. Thus, the expression of GGPP synthase is regulated in multiple tissues in obesity and is induced during adipocyte differentiation. Altered regulation in the synthesis of isoprenoids for protein prenylation in obesity might be a factor determining the ability of the cells to respond to hormonal stimulation requiring both Ras-related small GTPases and trimeric G protein-coupled receptors.

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Abstract 255: Cytoprotective Effects of Erythropoietin and Erythropoietin-derivatives in Ischaemic Human Myotubes and the Role of Pi3k/akt Signalling Pathway

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a255 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Rebekah Sian Hwee Yu ◽

Daryll Baker ◽

David Abraham ◽

Janice Tsui

Keyword(s):

Skeletal Muscle ◽

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Caspase 3 ◽

Signalling Pathway ◽

Critical Limb Ischaemia ◽

Human Myotubes ◽

Muscle Biopsies

Objectives Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously demonstrated the presence and interaction of the EpoR and βcR in human skeletal muscle. Here we aim to investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish a potential downstream signalling pathway utilised in protecting cells from apoptosis (including Jak-2, PI3k/Akt, NFkB). Methods Gastrocnemius muscle biopsies were obtained from patients with critical limb ischaemia and control samples were obtained from non-ischaemic patients. Human myoblasts were isolated from muscle biopsies, cultured, and allowed to differentiate into myotubes in order to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitors, LY294002 and wortmannin, were then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Following this, inhibitors against the upstreatm (Jak-2) and downstream (NFkB) molecules were also investigated. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis. Results Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of all inhibitors, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Conclusion The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.

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Abstract 234: Interleukin 2 Promotes Proliferation and Migration in Vascular Smooth Muscle Cells

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.234 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Spencer Barnhill ◽

Prakash Arumugam ◽

John Matsuura ◽

Scott Berceli ◽

Katie Carroll ◽

...

Keyword(s):

Smooth Muscle ◽

Protein Expression ◽

Western Blot ◽

Vascular Smooth Muscle Cells ◽

Blot Analysis ◽

Intimal Hyperplasia ◽

Western Blot Analysis ◽

Interleukin 2 ◽

And Migration ◽

Proliferation And Migration

Interleukin-2 (IL-2) is primarily known as a soluble cytokine that regulates T cell responses. We previously reported, however, that IL-2 is retained in the extracellular matrix by association with perlecan, a heparan sulfate proteoglycan (HSPG). Perlecan is the main HSPG in vascular basement membranes, and previous studies from our laboratory demonstrated that, in human arteries, vascular smooth muscle cells (VSMC) are surrounded by perlecan-bound IL-2. We also noted that IL-2 deficient mice lose SMCs with age, leading to widened esophagi and aortic aneurysms. Given this information, we hypothesized that IL-2 has a direct impact on VSMC, and that VSMC express functional IL-2 receptors (IL-2R). We therefore examined both protein and mRNA expression of each of the three IL-2R subunits (alpha, beta, gamma) on human VSMC grown from arterial explants. These VSMC expressed SMC actin, smooth muscle myosin heavy chain, and when quiescent, smoothelin. Protein expression was assessed by in cell Western and by Western blot analysis. Receptor expression was evaluated under distinct culture conditions, which yielded highly proliferative, intermediate, or quiescent VSMC. Contractile protein expression was low, intermediate, or high, respectively, consistent with the characteristics of proliferating vs quiescent SMCs. Each phenotype expressed all 3 subunits of the IL-2R. IL-2 subunits appeared to follow a cytoskeletal pattern in cells expressing high levels of contractile proteins. Western blot analysis of VSMC lysates revealed expression of all 3 receptors at molecular weights identical to lysates from a T cell line. VSMCs also expressed mRNA for each receptor subunit. Functionally, IL-2 promoted migration (using a Boyden chamber assay) and proliferation in a dose dependent fashion. Because excess proliferation and migration are critical to intimal hyperplasia, we asked whether IL-2 levels change under conditions known to generate intimal hyperplasia. In a rabbit model, IL-2 mRNA increased in venous grafts exposed to high flow for 2h. IL-2 levels, by Western blot, were also increased in human hyperplastic veins. In conclusion, these data show that VSMC have functional IL-2R, and suggest that IL-2 may contribute to the development of intimal hyperplasia.

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Specific detection of the endogenous transient receptor potential (TRP)-1 protein in liver and airway smooth muscle cells using immunoprecipitation and Western-blot analysis

Biochemical Journal ◽

10.1042/bj20020061 ◽

2002 ◽

Vol 364 (3) ◽

pp. 641-648 ◽

Cited By ~ 50

Author(s):

Hwei Ling ONG ◽

Jinglong CHEN ◽

Tim CHATAWAY ◽

Helen BRERETON ◽

Lei ZHANG ◽

...

Keyword(s):

Smooth Muscle ◽

Guinea Pig ◽

Western Blot ◽

Airway Smooth Muscle ◽

Blot Analysis ◽

Western Blot Analysis ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Airway Smooth Muscle Cells ◽

Transient Receptor

Although there are numerous reports of the presence of mRNA encoding the transient receptor potential (TRP)-1 protein in animal cells and of the detection of the heterologously expressed TRP-1 protein by Western-blot analysis, it has proved difficult to unequivocally detect endogenous TRP-1 proteins. A combination of immunoprecipitation and Western-blot techniques, employing a polyclonal antibody and a monoclonal antibody respectively, was developed. Using this technique, a band of approx. 80kDa was detected in extracts of H4-IIE rat liver hepatoma cell line and guinea-pig airway smooth muscle (ASM) cells transfected with human TRPC-1 cDNA. In extracts of untransfected H4-IIE cells, ASM cells, rat brain and guinea-pig brain, a band of approx. 92kDa was detected. Reverse transcriptase PCR experiments detected cDNA encoding both the α- and β-isoforms of TRP-1 in H4-IIE cells. Treatment of protein extracts with peptide N-glycosidase F indicated that the 92kDa band represents an N-glycosylated protein. Western blots conducted with a commercial polyclonal anti-(TRP-1) antibody (Alm) detected a band of 120kDa in extracts of H4-IIE cells and guinea-pig ASM cells. A combination of immunoprecipitation and Western-blotting techniques with the Alm antibody did not detect any bands at 92kDa or 120kDa in extracts of H4-IIE and ASM cells. It is concluded that (a) the 92-kDa band detected in untransfected H4-IIE and ASM cells corresponds to the N-glycosylated β-isoform of endogenous TRP-1, (b) the combined immunoprecipitation and Western-blot approach, employing two different antibodies, provides a reliable and specific procedure for detecting endogenous TRP-1 proteins, and (c) that caution is required in developing and utilizing anti-(TRP-1) antibodies.

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Inhibition of smooth muscle force generation by focal adhesion kinase inhibitors in the hyperplastic human prostate

AJP Renal Physiology ◽

10.1152/ajprenal.00011.2014 ◽

2014 ◽

Vol 307 (7) ◽

pp. F823-F832 ◽

Cited By ~ 14

Author(s):

Thomas Kunit ◽

Christian Gratzke ◽

Andrea Schreiber ◽

Frank Strittmatter ◽

Raphaela Waidelich ◽

...

Keyword(s):

Smooth Muscle ◽

Western Blot ◽

Muscle Contraction ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Blot Analysis ◽

Western Blot Analysis ◽

Smooth Muscle Contraction ◽

Human Prostate ◽

Focal Adhesion Proteins

Smooth muscle contraction may be critical for lower urinary tract symptoms (LUTS) in patients with benign prostate hyperplasia and requires stable anchorage of the cytoskeleton to the cell membrane. These connections are regulated by focal adhesion kinase (FAK). Here, we addressed the involvement of FAK in the regulation of smooth muscle contraction in hyperplastic human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Expression of FAK and focal adhesion proteins was assessed by Western blot analysis and immunohistochemical stainings. Effects of the FAK inhibitors PF-573228 and Y-11 on contraction of prostate strips were examined in the organ bath. Expression of FAK and focal adhesion proteins (integrin-5α, paxilin, and c-Src) was detected by Western blot analysis in prostate samples. By double immunofluorescence staining with calponin and pan-cytokeratin, expression of FAK was observed in stromal and epithelial cells. Immunoreactivity for FAK colocalized with integrin-5α, paxilin, talin, and c-Src. Stimulation of prostate tissues with the α1-adrenergic agonist phenylephrine increased the phosphorylation state of FAK at Tyr397 and Tyr925 with different kinetics, which was blocked by the α1-adrenoceptor antagonist tamsulosin. Norepinephrine and phenylephrine induced concentration-dependent contractions of prostate strips. Both FAK inhibitors PF-573228 and Y-11 significantly inhibited norepinephrine- and phenylephrine-induced contractions. Finally, PF-573228 and Y-11 inhibited contractions induced by electric field stimulation, which was significant at the highest frequency. In conclusion, α1-adrenergic smooth muscle contraction or its regulation involves FAK in the human prostate. Consequently, FAK may be involved in the pathophysiology of LUTS and in current or future LUTS therapies.

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Defining the Prion Type of Fatal Familial Insomnia

Pathogens ◽

10.3390/pathogens10101293 ◽

2021 ◽

Vol 10 (10) ◽

pp. 1293

Author(s):

Wiebke Jürgens-Wemheuer ◽

Arne Wrede ◽

Walter Schulz-Schaeffer

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Fatal Familial Insomnia ◽

Proteinase K ◽

Transmissible Spongiform Encephalopathies ◽

Valid Criterion ◽

Sporadic Cjd

Fatal familial insomnia (FFI) belongs to the genetic human transmissible spongiform encephalopathies (TSE), such as genetic Creutzfeldt-Jakob disease (CJD) or Gerstmann-Straeussler-Scheinker syndrome (GSS). Here, we analyzed the properties of the pathological prion protein in six FFI cases by Western blot analysis, a protein aggregate stability assay, and aggregate deposition characteristics visualized with the paraffin-embedded tissue blot. While in all cases the unglycosylated fragment in Western blot analysis shared the same size with sporadic CJD prion type 2, the reticular/synaptic deposition pattern of the prion aggregates resembled the ones found in sporadic CJD type 1 (CJD types according to the Parchi classification from 1999). Regarding the conformational stability against denaturation with GdnHCl, FFI prion aggregates resembled CJD type 1 more than type 2. Our results suggest that the size of the proteinase-K-resistant fragments is not a valid criterion on its own. Additional criteria supplying information about conformational differences or similarities need to be taken into account. FFI may resemble a prion type with its own conformation sharing properties partly with type 1 and type 2 prions.

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Abstract 1958: MicroRNA Mir-27b Rescues Impaired Angiogenic Function of Endothelial Progenitor Cells and Accelerates Wound Healing in Type 2 Diabetes

Circulation ◽

10.1161/circ.118.suppl_18.s_412 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Jie-Mei Wang ◽

Jun Tao ◽

Alex F Chen

Keyword(s):

Type 2 Diabetes ◽

Wound Healing ◽

Western Blot ◽

Real Time ◽

Blot Analysis ◽

Real Time Pcr ◽

Western Blot Analysis ◽

Type 2 Diabetic

Endothelial progenitor cells (EPCs) play a key role in angiogenesis, which is dysfunctional in diabetes. MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level. However, whether miRNAs regulate EPC-mediated angiogenesis in diabetes is unknown. We tested the hypothesis that mir-27b rescues impaired EPC angiogenesis in vitro and in vivo via suppressing anti-angiogenic molecule thrombospondin-1 (TSP-1) in type 2 diabetes. Bone marrow-derived EPCs from adult male (C57BLKS/J, 9 weeks) type 2 diabetic db/db and their normal littermates db/+ mice (glucose 371.8±37.8 vs. 167.5±21.3 mg/dL, n=38, p<0.05) were used. miRNA processing enzyme Dicer in EPCs was decreased by >40% in db/db vs. db/+ mice (Western blot analysis, n=4 p<0.01), paralleled with >66% reduction of mir-27b expression (real-time PCR, n=4, p<0.05). Both TSP-1 mRNA and protein in EPCs were significantly higher in db/db vs. db/+ mice (real-time PCR, 130.1%, n=4, p<0.05, Western blot analysis, 127.4%, n=4 p<0.05), which were suppressed upon mir-27b mimic transfection (by 75%, real-time PCR and 69%, Western blot analysis, n=4 – 6, p<0.01). EPC-induced angiogenesis was decreased by >70% in db/db vs. db/+ mice (Matrigel tube formation assay, n=4, p<0.05), which was rescued upon mir-27b mimic transfection or silencing TSP-1 expression by its siRNA (both n=4, p<0.05). Furthermore, inhibition of mir-27b in normal EPCs increased their TSP-1 protein by 117.5% (n=6, p<0.05) and impaired their angiogenesis by 81.5% (n=4, p<0.01), both were reversed by silencing TSP-1 expression by its siRNA. Finally, excisional wound closure was markedly delayed in db/db vs. db/+ mice (4-mm punch biopsy, n=4, p<0.05), accompanied by impaired wound angiogenesis (perfusion index by Laser Doppler, n=4, p<0.05). Cell therapy of diabetic EPCs (3×10 5 cells) transfected with mir-27b mimic onto diabetic wounds significantly accelerated their closure rates (n=4, p<0.05 vs. diabetic EPCs alone), with a concomitant augmentation of in vivo wound angiogenesis (n=4, p<0.05). Mir-27b rescues impaired EPC angiogenesis and accelerates wound healing in type 2 diabetic mice, at least in part, via suppressing TSP-1 expression. This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

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Validation of anti-glucocerebrosidase antibodies for western blot analysis on protein lysates of murine and human cells

Biochemical Journal ◽

10.1042/bcj20180708 ◽

2019 ◽

Vol 476 (2) ◽

pp. 261-274 ◽

Cited By ~ 4

Author(s):

Wenduo Qi ◽

Brad A. Davidson ◽

Matthew Nguyen ◽

Taylor Lindstrom ◽

Richard J. Grey ◽

...

Keyword(s):

Western Blot ◽

Blot Analysis ◽

Western Blot Analysis ◽

Lysosomal Storage ◽

Gene Encoding ◽

Custom Made ◽

Hydrolysis Of ◽

Immortalized Neurons ◽

Human Wild Type

Abstract Gaucher disease (GD) is a rare lysosomal storage disorder caused by mutations in the GBA1 gene, encoding the lysosome-resident glucocerebrosidase enzyme involved in the hydrolysis of glucosylceramide. The discovery of an association between mutations in GBA1 and the development of synucleinopathies, including Parkinson disease, has directed attention to glucocerebrosidase as a potential therapeutic target for different synucleinopathies. These findings initiated an exponential growth in research and publications regarding the glucocerebrosidase enzyme. The use of various commercial and custom-made glucocerebrosidase antibodies has been reported, but standardized in-depth validation is still not available for many of these antibodies. This work details the evaluation of several previously reported glucocerebrosidase antibodies for western blot analysis, tested on protein lysates of murine gba+/+ and gba−/− immortalized neurons and primary human wild-type and type 2 GD fibroblasts.

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Autophagy and Mitophagy Promotion in a Rat Model of Endometriosis

International Journal of Molecular Sciences ◽

10.3390/ijms22105074 ◽

2021 ◽

Vol 22 (10) ◽

pp. 5074

Author(s):

Rosalba Siracusa ◽

Ramona D’Amico ◽

Daniela Impellizzeri ◽

Marika Cordaro ◽

Alessio Filippo Peritore ◽

...

Keyword(s):

Western Blot ◽

Rat Model ◽

Blot Analysis ◽

Western Blot Analysis ◽

Mammalian Target Of Rapamycin ◽

Immunohistochemical Analysis ◽

Reproductive Age ◽

Serine Threonine Kinase ◽

Autophagy Pathway ◽

The Impact

Endometriosis is a gynecological condition affecting patients in reproductive age. The aim of this paper was to assess the effects of the autophagy and mitophagy induction in a rat model of endometriosis. Endometriosis was induced by the injection of uterine fragments, and rapamycin (0. 5 mg/kg) was administered once per week. One week from the induction, rats were sacrificed, and laparotomy was performed to collect the endometriotic implants and to further process them for molecular analysis. Western blot analysis was conducted on explanted lesions to evaluate the autophagy pathway during the pathology. Elevated phospho-serine/threonine kinase (p-AKT) and mammalian target of rapamycin (mTOR) expressions were detected in vehicle-treated rats, while Beclin and microtubule-associated protein 1A/1B-light chain 3 II (LC3II) expressions were low. Additionally, samples collected from vehicle groups indicated low Bnip3, Ambra1, and Parkin expressions, demonstrating impaired autophagy and mitophagy. Rapamycin administration reduced p-AKT and mTOR expressions and increased Beclin and LC3II, Bnip3, Ambra1, and Parkin expressions, activating both mechanisms. We also evaluated the impact of the impaired autophagy and mitophagy pathways on apoptosis and angiogenesis. Rapamycin was administered by activating autophagy and mitophagy, which increased apoptosis (assessed by Western blot analysis of Bcl-2, Bax, and Cleaved-caspase 3) and reduced angiogenesis (assessed by immunohistochemical analysis of vascular endothelial grow factor (VEGF) and CD34) in the lesions. All of these mechanisms activated by the induction of the autophagy and mitophagy pathways led to the reduction in the lesions’ volume, area and diameter.

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