Search results for " Western"
showing 10 items of 687 documents
Requirement of Retinoic Acid Receptor Isotypes α, β, and γ during the Initial Steps of Neural Differentiation of PCC7 Cells
2005
Retinoic acid (RA) is indispensable for morphogenesis and differentiation of several tissues, including the nervous system. The requirement of the RA receptor (RAR) isotypes alpha, beta, and gamma and the putative role of retinoid X receptor-(RXR) signaling in RA-induced neural differentiation, was analyzed. For this compound-selective retinoids and the murine embryonal carcinoma cell line PCC7, a model system for RA-dependent neural differentiation was used. The present paper shows that proliferating PCC7 cells primarily express RXRalpha and RARalpha, lower levels of RXRbeta, and barely detectable amounts of RARbeta, RARgamma, and RXRgamma. At receptor-selective concentrations, only a RARa…
Antagonistic feedback loops involving Rau and Sprouty in the Drosophila eye control neuronal and glial differentiation.
2013
During development, differentiation is often initiated by the activation of different receptor tyrosine kinases (RTKs), which results in the tightly regulated activation of cytoplasmic signaling cascades. In the differentiation of neurons and glia in the developing Drosophila eye, we found that the proper intensity of RTK signaling downstream of fibroblast growth factor receptor (FGFR) or epidermal growth factor receptor required two mutually antagonistic feedback loops. We identified a positive feedback loop mediated by the Ras association (RA) domain-containing protein Rau that sustained Ras activity and counteracted the negative feedback loop mediated by Sprouty. Rau has two RA domains t…
Hamster Bcl-2 Protein Is Cleaved in Vitro and in Cells by Caspase-9 and Caspase-3
2001
Full-length cDNA of hamster bcl-2 (771 nt) was cloned by RT-PCR and inserted into pGEX-4T-1 to produce the recombinant hamster Bcl-2 protein. The purified recombinant Bcl-2 protein (26.4 kDa) was used as a substrate for the active human caspase-3 and caspase-9 in vitro. It is shown here that Bcl-2 is efficiently cleaved by caspase-3 to a 23 kDa fragment. Although not possessing a putative caspase-9 cleavage site in its sequence, hamster Bcl-2 was also cleaved by caspase-9 into exactly the same 23 kDa cleavage product, indicating that cleavage occurred at the same site. Caspase-3- and caspase-9-mediated cleavage of Bcl-2 was efficiently blocked by caspase-3 (zDEVD) and caspase-9 (zLEHD) inhi…
Probing human beta1- and beta2 -adrenoceptors with domain-specific fusion protein antibodies.
1997
In order to generate antibodies suitable for immunological studies on beta-adrenoceptors constitutively expressed at low levels in cells or tissues we have produced fusion proteins of the amino- and carboxy-terminus, and the second extracellular loop of the human beta 1- or beta 2-adrenoceptors with bacterial glutathione-S-transferase in E. coli. Rabbit antibodies raised against these fusion proteins strongly reacted with intact human beta 1- or beta 2-adrenoceptors in a subtype- and domain-specific manner. Antibodies directed against the second extracellular loop of the beta 1-adrenoceptor reacted stronger with non-denatured receptors and decreased the affinity of the 3H-labelled antagonis…
Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor–avidin fusion protein
2003
The very high binding affinity of avidin to biotin is one of the highest to occur in nature. We constructed a fusion protein composed of avidin and the endocytotic LDL receptor in order to target biotinylated molecules to cells of the desired tissues. In addition to the native avidin, charge-mutated and nonglycosylated avidins were utilized as part of the fusion proteins, in order to modify its properties. All of the fusion protein versions retained the biotin-binding capacity. Although the specificity was not increased, however, fusion proteins composed of natural avidin and nonglycosylated avidin bound most efficiently to the biotinylated ligands. Fluorescence microscopy and atomic force …
The (2'-5')Oligoadenylate Synthetase is Present in the Lowest Multicellular Organisms, the Marine Sponges. Demonstration of the Existence and Identif…
1995
We have proved the presence of (2'-5')oligoadenylates [(2'-5')An] and oligoadenylate synthetase [(2'-5')An synthetase] in the marine sponge Geodia cydonium. (2'-5')An isolated from sponge crude extract competed with authentic (2'-5')An for binding to polyclonal antiserum against (2'-5')An. HPLC analysis revealed the presence of nucleotides eluting with molecular markers for (2'-5')A oligomers. The biological activity of sponge (2'-5')An was demonstrated by inhibiting the protein biosynthesis in rabbit reticulocyte lysate. The activity of the (2'-5')An synthetase, present in crude sponge extract, was found to be high compared to that in mammalian interferon-treated cell extract. The (2'-5')A…
Enrichment of Retinal Ganglion Cells in Rat Retinal Lysate by Excimer Laser Ablation of the Outer Retina
2013
PURPOSE. Retinal ganglion cells (RGC) are a relatively small cell population in the retina. This leads to an unfavorable signal-tonoise ratio when analyzing RGC proteins in whole retina lysate. We present a novel technique to obtain RGC-enriched rat retinal lysate by removing the outer retinal layers with an excimer laser before lysation. METHODS. Outer retinal layers were ablated with an excimer laser on flat mounted retinas from adult albino rats. 4 0 6Diamidino-2-phenylindole dihydrochloride hydrate (DAPI) nuclear staining was used to assess the ablation efficacy (n ! 6). Western blot for layer specific markers (rhodopsin, parvalbumin, b-III-tubulin) was performed to quantify changes in …
SANS (USH1G) expression in developing and mature mammalian retina
2008
AbstractThe human Usher syndrome (USH) is the most common form of combined deaf-blindness. Usher type I (USH1), the most severe form, is characterized by profound congenital deafness, constant vestibular dysfunction and prepubertal-onset of retinitis pigmentosa. Five corresponding genes of the six USH1 genes have been cloned so far. The USH1G gene encodes the SANS (scaffold protein containing ankyrin repeats and SAM domain) protein which consists of protein motifs known to mediate protein–protein interactions. Recent studies indicated SANS function as a scaffold protein in the protein interactome related to USH.Here, we generated specific antibodies for SANS protein expression analyses. Our…
Differential expression and interaction with the visual G-protein transducin of centrin isoforms in mammalian photoreceptor cells.
2004
Photoisomerization of rhodopsin activates a heterotrimeric G-protein cascade leading to closure of cGMP-gated channels and hyperpolarization of photoreceptor cells. Massive translocation of the visual G-protein transducin, Gt, between subcellular compartments contributes to long term adaptation of photoreceptor cells. Ca(2+)-triggered assembly of a centrin-transducin complex in the connecting cilium of photoreceptor cells may regulate these transducin translocations. Here we demonstrate expression of all four known, closely related centrin isoforms in the mammalian retina. Interaction assays revealed binding potential of the four centrin isoforms to Gtbetagamma heterodimers. High affinity b…
A Candida albicans 37 kDa polypeptide with homology to the laminin receptor is a component of the translational machinery.
1998
A cDNA encoding a 37 kDa protein was isolated from an expression library using antibodies raised against mycelial cell walls fromCandida albicans.The 37 kDa protein has over 60% sequence identity with the 37 kDa laminin-binding protein (LBP) from humans and over 80% identity with the Yst proteins ofSaccharomyces cerevisiae. TheC. albicansprotein was named CaYst1. It was found in membrane and ribosome fractions but surprisingly, was not found in cell walls. Unlike the human LBP, CaYst1p does not bind laminin. These data indicate that CaYst1p is not a cell-surface receptor for laminin as has been proposed for the human LBP. Instead, like theS. cerevisiaeYst proteins, it appears to be a riboso…