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Western Immunoblot Analysis (western + immunoblot_analysis)
Selected AbstractsEffects of Secreted Frizzled-Related Protein 3 on Osteoblasts In Vitro,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2004Yoon-Sok Chung Abstract To examine if sFRP3s act as decoy receptors for Wnt, we examined the effects of recombinant sFRP3 on mouse osteoblast proliferation and differentiation. We found that sFRP3 unexpectedly increased osteoblast differentiation, suggesting it may act through other mechanisms besides acting as a decoy receptor for Wnt's. Introduction: Secreted frizzled-related proteins (sFRPs) are a truncated form of frizzled receptor, missing both the transmembrane and cytosolic domains. Because previous studies have shown that sFRPs bind and act as decoy receptors for Wnt proteins that promote osteoblast differentiation, we postulated that sFRP3 acts as an inhibitor of osteoblast differentiation. Materials and Methods: We examined the effects of mouse recombinant sFRP3 and/or Wnt-3A on cell proliferation and differentiation using MC3T3-E1 mouse osteoblasts and primary cultures of mouse bone marrow stromal cells. We evaluated the effects of sFRP3 on ,-catenin levels using Western immunoblot analyses. Results: We found that sFRP3 suppressed osteoblast cell number in a dose-dependent manner that was the result of a decrease in proliferation and not because of an increase in apoptosis. Surprisingly, sFRP3 increased osteoblast differentiation, which could not be explained based on sFRP3 acting as a decoy receptor for stimulatory Wnt's. Furthermore, sFRP3 did not inhibit Wnt3A-induced increase in alkaline phosphatase (ALP) activity. Wnt3A, but not sFRP3 treatment, increased cellular ,-catenin levels, and sFRP3 failed to block Wnt3A-induced increase in cellular ,-catenin levels. Treatment with endostatin, an agent known to degrade ,-catenin, did not inhibit sFRP3-induced increase in ALP activity. sFRP1, like sFRP3, inhibited proliferation and stimulated ALP activity in MC3T3-E1 mouse osteoblasts. Conclusions: Based on our findings, we conclude that sFRP3 decreased osteoblast proliferation and unexpectedly increased parameters of osteoblast differentiation. Based on our findings, we propose that sFRP3 may stimulate differentiation through a ,-catenin-independent pathway in addition to its previously known function as a decoy receptor for Wnt's. [source] Thyroglobulin type-1 domain protease inhibitors exhibit specific expression in the cortical ooplasm of vitellogenic rainbow trout oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2004Antony W. Wood Abstract The synthesis, uptake, and processing of yolk proteins remain poorly described aspects of oviparous reproductive development. In this study, we report the identification and characterization of two protease inhibitors in rainbow trout ovary whose expression and distribution are directly associated with yolk protein uptake in vitellogenic oocytes. The first transcript, termed "oocyte protease inhibitor-1" (OPI-1), is predicted to encode a 9.1 kDa, 87 amino acid protein containing a single thyroglobulin type-1 (TY) domain, identifying it as a putative TY domain inhibitor. The second transcript, termed OPI-2, is predicted to encode an 18.3 kDa, 173 amino acid protein with two similar, but not identical, TY domains. Messenger RNA expression of both genes was first detected in ovarian tissues at the onset of vitellogenesis, and persisted throughout the vitellogenic growth phase. We did not detect expression of either gene in previtellogenic ovaries, nor in any somatic tissues examined. Expression of OPI-1 mRNA was significantly reduced in atretic follicles as compared to healthy vitellogenic follicles, suggesting a downregulation of inhibitor expression during oocyte atresia. Western immunoblot analyses of whole yolk from vitellogenic oocytes revealed the presence of two immunoreactive proteins that corresponded to the predicted sizes of OPI-1 and OPI-2. We detected strong crossreactivity of this antiserum with specific vesicles in the cortical ooplasm of vitellogenic oocytes, in regions directly associated with vitellogenin processing. The identification of OPI-1 and OPI-2 provides new evidence for the expression of multiple TY domain protease inhibitors likely involved in the regulation of yolk processing during oocyte growth in salmonids. Mol. Reprod. Dev. 69: 205,214, 2004. © 2004 Wiley-Liss, Inc. [source] Endochitinase activity in the apoplastic fluid of Phellinus weirii -infected Douglas-fir and its association with over wintering and antifreeze activityFOREST PATHOLOGY, Issue 5 2003A. Zamani Summary Extracellular proteins were extracted from Phellinus weirii infected Douglas-fir (Pseudotsuga menziesii var. menziesii) roots and needles to examine endochitinase activity. Chitinases have been associated with the plant's defence response against fungal attack because they hydrolyse chitin, a structural component of fungal cell walls. Protein separation using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western immunoblot analysis using a polyclonal antibody specific to an endochitinase-like protein (ECP) resulted in the detection of up to three polypeptides between 27 and 30 kDa in size. Two-dimensional gel electrophoresis (2-D PAGE) followed by Western immunoblot analysis revealed that the apoplastic fluid contained multiple ECP isoforms with isoelectric points (pIs) ranging from 5.3 to 5.8 and molecular masses of 27,30 kDa. Chitinase activity in needle and root tissues was measured spectrophotometrically using a colorimetric assay. A gel overlay technique using glycol chitin as a substrate for endochitinase was applied to confirm that the ECP antibody detected an enzymatically active protein. The apoplastic fluid collected from P. weirii -infected winter Douglas-fir needles showed anti-freeze activity and seasonal analysis of needle tissue showed some evidence of ECP accumulation in winter months. ECP was distributed systemically throughout the tree. Increased levels of endochitinase activity in the region of P. weirii infection supports a physiological role for ECP in the plant defence response. Résumé Les protéines extra-cellulaires ont été extraites des racines et aiguilles de douglas (Pseudotsuga menziesii var menziesii) infectés par Phellinus weirii (Murr.) Gilbn., pour étudier l'activité endochitinase. Les chitinases ont été associées aux réactions de défense des plantes contre les attaques fongiques parce-qu'elles hydrolysent la chitine, un composant de la paroi des cellules fongiques. La séparation des protéines, réalisée par électrophorèse en gel de polyacrylamide avec sodium dodecyl sulfate (SDS-PAGE), suivie par une analyse par Western immunoblot en utilisant un anticorps polyclonal spécifique d'une protéine de type endochitinase (ECP), a permis la détection de 3 polypeptides de taille comprise entre 27 et 30 kDa. Une électrophorèse sur gel en 2-dimensions (2-D PAGE) suivie par une analyse par Western immunoblot a révélé que le fluide apoplastique contient de multiples isoformes d'ECP avec des pI dans une gamme de 5.3 à 5.8 et des masses moléculaires de 27 à 30 kDa. L'activité chitinase dans les aiguilles et tissus racinaires a été mesurée par spectrophotométrie par une méthode colorimétrique. Une technique d'overlay utilisant de la chitine glycol comme substrat de l'endochitinase a été appliquée pour confirmer que l'anticorps ECP avait détecté une protéine active du point de vue enzymatique. Le fluide apoplastique d'aiguilles récoltées en hiver sur des douglas infectés par P. weirii a montré une activité antigel et l'analyse saisonnière des tissus foliaires a montré une certaine accumulation d'ECP pendant l'hiver. L'ECP est répartie de façon systémique dans l'ensemble de l'arbre. Les niveaux accrus d'activité endochitinase dans la zone infectée par P. weirii suggère un rôle physiologique de l'ECP dans les réactions de défense de la plante. Zusammenfassung Aus Wurzeln und Nadeln von mit Phellinus weirii infizierten Douglasien (Pseudotsuga menziesii var. menziesii) wurden extrazelluläre Proteine extrahiert, um die Endochitinase-Aktivität zu bestimmen. Chitinasen werden mit der pflanzlichen Abwehrreaktion auf Pilzinfektionen in Verbindung gebracht, da sie Chitin, eine Strukturkomponente der pilzlichen Zellwand, hydrolysieren. Die Proteine wurden mit Natrium-Dodecyl-Sulfat-Polyacrylamid-Gelelektrophorese (SDS-PAGE) getrennt, gefolgt von einer Western Immunoblot-Analyse mit einem gegen ein Endochitinase-ähnliches Protein (ECP) spezifischen polyklonalen Antikörper. Hiermit liessen sich bis zu drei Polypeptide zwischen 27-30 kDa nachweisen. Eine zweidimensionale Gelelektrophorese (2-D PAGE) mit anschliessender Western Immunoblot-Analyse ergab, dass die Apoplastenflüssigkeit multiple ECP-Isoformen enthielt (mit pIs von 5,3 bis 5,8 und Molekularmassen von 27 bis 30 kDa). Die Chitinase-Aktivität wurde auch im Nadel- und Wurzelgewebe spektrophotometrisch mit einer Farbreaktion gemessen. Um sicher zu stellen, dass der ECP-Antikörper ein enzymatisch aktives Protein nachwies, wurde eine Gel-Overlay-Methode verwendet, mit Glycolchitin als Substrat für die Endochitinase. Die Apoplastenflüssigkeit der Nadeln von mit P. weirii infizierten Douglasien zeigte in Winterzustand eine Antifrost-Aktivität, ihre Analyse während des gesamten Jahres ergab aber keine Hinweise auf eine ECP-Anreicherung während der Wintermonate. ECP war systemisch im gesamten Baum enthalten. Die erhöhte Endochitinase-Aktivität in Bereichen mit P. weirii -Infektion lässt auf eine physiologische Rolle von ECP in der Pflanzenabwehr schliessen. [source] MicroRNA-34a is an important component of PRIMA-1-induced apoptotic network in human lung cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 2 2010Wenrui Duan Abstract Restoration of p53 function in tumor cells would be an attractive strategy for lung cancer therapy because p53 mutations are found in more than 50% of lung cancers. The small molecule PRIMA-1 has been shown to restore the tumor suppression function of p53 and to induce apoptosis in human tumor cells. The mechanism of apoptosis induced by PRIMA-1 remains unclear. We investigated the effects of PRIMA-1 in apoptosis with Western immunoblot analysis, TaqMan microRNA real-time PCR, cell viability analysis and flow cytometry using human lung cancer cell lines containing mutant (H211 and H1155), wild-type (A549) or null (H1299) p53. PRIMA-1 induced massive apoptosis in the H211 and H1155 cells, but was less toxic to the A549 and H1299 cells. Western immunoblot analysis showed cleavage of PARP in H211 and H1155 cells but not in A549 and H1299 cells following treatment with PRIMA-1. In addition, p53 protein was also phosphorylated in H211 and H1155 cells. TaqMan microRNA assay showed that the expression of microRNA-34a was increased in the H211 and H1155 cells posttreatment. Knockdown microRNA-34a decreased the rate of apoptosis caused by PRIMA-1. The above results suggest that microRNA-34a is one of the important components of PRIMA-1-induced apoptotic network in the cancer cells harboring mutant p53. [source] Insulin-like growth factor-I ameliorates demyelination induced by tumor necrosis factor-, in transgenic miceJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2007Ping Ye Abstract Our groups have reported that tumor necrosis factor-, (TNF-,) causes myelin damage and apoptosis of oligodendrocytes and their precursors in vitro and in vivo. We also have reported that insulin-like growth factor-I (IGF-I) can protect cultured oligodendrocytes and their precursors from TNF-,-induced damage. In this study, we investigated whether IGF-I can protect oligodendrocytes and myelination from TNF-,-induced damage in vivo by cross-breeding TNF-, transgenic (Tg) mice with IGF-I Tg mice that overexpress IGF-I exclusively in brain. At 8 weeks of age, compared with those of wild-type (WT) mice, the brain weights of TNF-, Tg mice were decreased by ,20%, and those of IGF-I Tg mice were increased by ,20%. The brain weights of mice that carry both TNF-, and IGF-I transgenes (TNF-,/IGF-I Tg mice) did not differ from those of WT mice. As judged by histochemical staining and immunostaining, myelin content in the cerebellum of TNF-,/IGF-I Tg mice was similar to that in WT mice and much more than that in TNF-, Tg mice. Consistently, Western immunoblot analysis showed that myelin basic protein (MBP) abundance in the cerebellum of TNF-,/IGF-I Tg mice was double that in TNF-, Tg mice. In comparison with WT mice, the number of oligodendrocytes was decreased by ,36% in TNF-, Tg mice, whereas it was increased in IGF-I Tg mice by ,40%. Oligodendrocyte number in TNF-,/IGF-I Tg mice was almost twice that in TNF-, Tg mice. Furthermore, IGF-I overexpression significantly reduced TNF-,-induced increases in apoptotic cell number, active caspase-3 abundance, and degradaion of MBP. Our results indicate that IGF-I is capable of protecting myelin and oligodendrocytes from TNF-,-induced damage in vivo. © 2007 Wiley-Liss, Inc. [source] Salivary immunoglobulin A directed to oral microbial GroEL in patients with periodontitis and their potential protective roleMOLECULAR ORAL MICROBIOLOGY, Issue 5 2006M. Fukui The aim of this study was to identify salivary immunoglobulin A (IgA) directed to oral microbial GroEL in patients with periodontitis and to demonstrate their potential protective role through a reduction of inflammatory cytokine production induced by microbial GroEL. Using five different proteins belonging to the heat-shock protein 60 family, Western immunoblot analysis of salivary IgA from 63 subjects revealed immunoreactivities with Campylobacter rectus GroEL and Porphyromonas gingivalis GroEL in subjects with periodontitis (P < 0.05) compared to control subjects. Using the BIACORE 1000 to measure the salivary IgA titers directed towards C. rectus GroEL, high resonance unit (RU) values were observed in the saliva samples from patients with periodontitis (P < 0.01). Furthermore, the number of teeth with deep pocket depth (,5 mm) showed a high correlation coefficient with the RU value (r = 0.50, P < 0.01). C. rectus GroEL possessed the ability to stimulate the production of interleukin-6 by gingival fibroblasts. Interestingly, salivary IgA antibody directed to C. rectus GroEL caused a partial inhibition of interleukin-6 production. This study showed a relationship between high levels of salivary IgA directed to GroEL and periodontal disease severity. Although additional investigations are required, salivary IgA to GroEL may have a protective role by reducing the inflammatory response induced by GroEL derived from periodontopathogenic bacteria. [source] Possible role of the protein kinase C/CPI-17 pathway in the augmented contraction of human myometrium after gestationBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2003Hiroshi Ozaki Activation of protein kinase C (PKC) by phorbol 12,13-dibutylate (PDBu, 1 ,M) induced sustained contractions with no increase in [Ca2+]i in nonpregnant and pregnant human myometria. The contractile effects of PDBu in pregnant myometrium were much greater than those in nonpregnant myometrium, and the contractions in pregnant myometrium were accompanied by an increase in myosin light chain (MLC) phosphorylation at Ser19. The contraction induced by PDBu in pregnant myometrium was inhibited by the inhibitors of conventional PKC isoforms, bisindolylmaleimides and indolocarbazole, such as Go6976, Go6983, and Go6850 (1 ,M). LY333531 (1 ,M), a specific inhibitor of PKC,, also inhibited the PDBu-induced contraction in the pregnant myometrium. In the pregnant myometrium permeabilized with , -toxin, PDBu increased the contractions induced at fixed Ca2+ concentration (0.3 ,M) both in nonpregnant and pregnant myometria, indicating Ca2+ sensitization of contractile elements. Western immunoblot analysis indicated that pregnant myometrium contained PKC isozymes such as conventional PKC (,, ,, ,), novel PKC (,, ,, ,), and atypical PKC (, but not , and ,). RT-PCR and real-time RT-PCR analysis indicated that, among the conventional PKC, the levels of mRNA of , isoform in pregnant human myometrium were greater than those in nonpregnant myometrium. CPI-17 is a substrate for PKC, and the phosphorylated CPI-17 is considered to inhibit myosin phosphatase. The levels of CPI-17 mRNA and protein expression were also greater in the pregnant myometrium. These results suggest that the PKC-mediated contractile mechanism is augmented in human myometrium after gestation, and that this augmentation may be attributable to the increased activity of the , PKC isoform and CPI-17. British Journal of Pharmacology (2003) 140, 1303,1312. doi:10.1038/sj.bjp.0705552 [source] VCP (p97) Regulates NFKB Signaling Pathway, Which Is Important for Metastasis of Osteosarcoma Cell LineCANCER SCIENCE, Issue 3 2002Tatsuya Asai In order to identify genes associated with metastasis, suppression subtractive hybridization (SSH) was performed using murine osteosarcoma cell line Dunn and its subline with higher metastatic potential, LM8. SSH revealed expression of the gene encoding valosin-containing protein (VCP; also known as p97) to be constitutively activated in LM8 cells, but it declined in Dunn cells when the cells became confluent. Because VCP is known to be involved in the ubiquitination process of Inhibitor-,B, (I,B,), an inhibitor of nuclear factor-,B (NF,B), whether VCP influences NF,B activation or not was examined by using VCP-transfected Dunn cells (Dunn/VCPs). When stimulated with tumor necrosis factor-, (TNF,), Dunn/VCPs showed constantly activated NF,B, although in the original Dunn cells and control vector transfectant (Dunn/Dunn-c) NF,B activation ceased when the cells became confluent. Western immunoblot analysis showed an increase of phosphorylated I,B, (p-IKB,) in the cytoplasm of confluent Dunn/Dunn-c cells compared to that of Dunn/VCPs. Therefore, decrease of p-IKB, degrading activity might be responsible for the decrease in NFKB activation. In vitro apoptosis assay demonstrated increased apoptosis rates of Dunn/Dunn-c cells after TNF, stimulation compared to those of Dunn/VCPs and LM8 cells. In vivo metastasis assay showed increased incidences of metastatic events in Dunn/VCP-1 inoculated male C3H mice compared to those in Dunn/Dunn-c inoculated mice. These findings suggested that VCP expression plays an important role in the metastatic process. Anti-apoptotic potential in these cells owing to constant NFKB activation via efficient cytoplasmic p-IKB, degrading activity may explain the increased metastatic potential of these cells. [source] | |||||||||||||