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Immunoreactive Bands (immunoreactive + bands)
Selected AbstractsMolecular cloning and expression of Tenebrio molitor ultraspiracle during metamorphosis and in vivo induction of its phosphorylation by 20-hydroxyecdysoneINSECT MOLECULAR BIOLOGY, Issue 3 2000M. Nicolaï Abstract Using a RT-PCR approach, the Tenebrio molitor homologue of Drosophila Ultraspiracle (TmUSP) was characterized. Its DNA binding domain shows a degree of identity with those of the other insect USPs. However, the ligand binding domain is closer to those of retinoid X receptors. Using an antibody raised against DmUSP, Western blot analysis of proteins from epidermis and other tissues revealed five immunoreactive bands, corresponding to different phosphorylated forms of a unique polypeptide, as shown by ,-phosphatase treatment. The nuclear form of TmUSP seems unphosphorylated. An in vivo 20-hydroxyecdysone treatment increases considerably and rapidly the phosphorylated forms of TmUSP. This post-translational modification may play a role in the 20-hydroxyecdysone response. [source] Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiationTHE JOURNAL OF PHYSIOLOGY, Issue 23 2008Dina Simkin TREK-2 expressed in mammalian cells exhibits small (,52 pS) and large (,220 pS) unitary conductance levels. Here we tested the role of the N-terminus (69 amino acids long) in the control of the unitary conductance, and role of the alternative translation initiation as a mechanism that produces isoforms of TREK-2 that show different conductance levels. Deletion of the first half (,1,36) of the N-terminus had no effect. However, deletion of most of the N-terminus (,1,66) resulted in the appearance of only the large-conductance channel (,220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants ,1,44 and ,1,54 produced ,90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at ,54 kDa and ,60 kDa from cells expressing wild-type TREK-2 that has three potential translation initiation sites (designated M1M2M3) within the N-terminus. Mutation of the second and third initiation sites from Met to Leu (M1L2L3) produced only the ,60 kDa isoform and the small-conductance channel (,52 pS). Mutants designed to produce translation from the second (M2L3) or third (M3) initiation site produced the ,54 kDa isoform, and the large conductance channel (,185,224 pS). M1L2L3, M2L3 and M3 were relatively selectively permeable to K+, as judged by the 51,55 mV shifts in reversal potential following a 10-fold change in [K+]o. PNa/PK values were also similar for M1L2L3 (,0.02), M2L3 (,0.02) and M3 (,0.03). Arachidonic acid, proton and membrane stretch activated, whereas dibutyryl-cAMP inhibited all three isoforms of TREK-2, indicating that deletion of the N-terminus does not abolish modulation. These results show that the small and large conductance TREK-2 channels are produced as a result of alternative translation initiation, producing isoforms with long and short N-termini, and that the distal half of the N-terminus controls the unitary conductance. [source] Detection and Localization of Two Constitutive NOS Isoforms in Bull SpermatozoaANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2003H. Meiser Summary Bull spermatozoa were examined for the presence and localization of constitutive Nitric Oxide Synthase (NOS), as nitric oxide (NO) is involved in calcium-dependent capacitation. In bull spermatozoa, NO generation is enhanced by l -arginine (3 ,m) and abolished by the NOS-inhibitor N -nitro- l -arginine methyl ester (l -NAME). In addition, presence of NOS in bull spermatozoa was verified by immunohistochemistry, revealing the existence of both neuronal NOS (nNOS) and endothelial NOS (eNOS) immunoreaction. These findings were confirmed by Western blot technique, showing immunoreactive bands at 161 kDa (nNOS) and 133 kDa (eNOS). Confocal laser microscopy localized nNOS related immunofluorescence at the acrosome cap of sperms and their flagellum-mainpart. This technique also identified eNOS staining spread over the spermatozoan head. In conclusion, immunohistochemistry, Western blot technique, and NO generation suggest the presence of n- and eNOS in bull spermatozoa. [source] Transient transfection of epidermal growth factor receptor gene into MCF7 breast ductal carcinoma cell lineCELL BIOCHEMISTRY AND FUNCTION, Issue 3 2005Majed S. Alokail Abstract Epidermal growth factor receptor (EGFR) is activated by autocrine growth factors in many types of tumours, including breast tumours. This receptor has been linked to a poor prognosis in breast cancer and may promote proliferation, migration, invasion, and cell survival as well as inhibition of apoptosis. Human breast ductal carcinoma MCF7 cells were transfected using FuGENEÔ 6 with 1,,g of pcDNA3-EGFR containing the full-length human EGFR promoter or 1,,g of the vectors alone (pcDNA3). The transfected cells were transferred into a 25-cm2 flask containing growth medium and G418. Confluent cultures were lysed, total protein levels measured and electrophoresed. The electrophoresed samples were transferred to nitrocellulose and incubated overnight at 4°C with either anti-EGFR or anti-phospho-ERK and immunoreactive bands were visualized using HRP-linked secondary antibody. We created a model system of EGFR overexpression in MCF7 clones with stably transfected pcDNA3/EGFR plasmid. These cells have been shown to promote substantial phosphorylation of both ERK1 and ERK2. The high level of EGFR and ERK1/2 phosphorylation was not seen in the pcDNA3 vector control cells or in non-transfected cells. In this article we describe successful transient transfection experiments on MCF7 cells using the FuGENEÔ 6 Transfection Reagent. The overexpression of EGFR could be a mediated stress response and a survival signal that involves ERK1 and ERK2 phosphorylation. Copyright © 2004 John Wiley & Sons, Ltd. [source] | ||||||||||