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BX50 Upright microscope, equipped with Color View camera (Soft Imaging System, Munster, Germany). ?For EM analyses, naive and allogeneic interacting subclones were fixed in 2.5 glutaraldehyde in seawater and stored at 4uC for 10 days. Next, they were washed thrice in 0.2M cacodylate buffer (pH 7.2?.4) and postfixed by 0.1 OsO4 in the same buffer for 1 h, at room temperature. After three buffer w
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Ligase activity. To test this, we expressed all of the Drosophila Roc proteins and chimeras as GST fusion proteins in E. coli and purified them (Fig. 6A). We then tested them for E3 ligase activity using a previously described in vitro assay that detects E2- and GST-Roc-dependent polyubiquitin formation in the absence of either Cullin or a particular substrate [24,26]. The ANBR protein was fully f
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Nally substitute for Roc2. We tested this by introducing a Roc1a transgene into the Roc2KG mutant background. Even in this genotype, Roc1a bound to Cul1 but not detectably to Cul5 (Fig. 8). Moreover, as we show in Fig. 6G, the pool of Cul5 available for binding Roc1a is greatly reduced in Roc2 mutant animals relative to wild type. Thus, the Cul1 and Cul5 E3 ligase complexes form independently of o
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Nally substitute for Roc2. We tested this by introducing a Roc1a transgene into the Roc2KG mutant background. Even in this genotype, Roc1a bound to Cul1 but not detectably to Cul5 (Fig. 8). Moreover, as we show in Fig. 6G, the pool of Cul5 available for binding Roc1a is greatly reduced in Roc2 mutant animals relative to wild type. Thus, the Cul1 and Cul5 E3 ligase complexes form independently of o
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To 40,000 units/L) in order to check impacts on Botryllus heart beat rates. For this experiment, we chose actively filtering colonies. Heparin treatments were carried out in Petri dishes containing 50 ml of seawater at 20uC. Ten minutes after heparin administration, the medium was replaced with fresh seawater. Heartbeats per 10 seconds were counted every five minutes for the same heart.Light and e
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Both of which are viable. The Roc2KG P element insertion is within the large intron, while the Roc2pBac piggyBac insertion is in a smaller intron upstream of the first protein-coding exon (Fig. 7A). RT-PCR analysis of embryonic mRNA (from mating homozygous males and virgin females) revealed that the Roc2KG allele expresses no detectable mRNA, while the Roc2pBac allele expresses a reduced amount of
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The nonself-recognizing cascade, prophenoloxidase system, with the oxygen carrier hemocyanin functioning as a substitute for prophenol oxidase. This hemolymph coagulation system is strongly activated by bacterial elicitors, in contrast to vertebrate coagulation where induction relies primarily on signals from damaged endothelial tissue. It is also evident that invertebrates like arthropods use dif
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We used laboratory-bred B. schlosseri colonies, offspring of founders collected at Monterey, Half Moon Bay and Moss Landing, CA, USA, and Banyuls, France marinas. The animals were maintained on glass slides at 20uC, in a flow-through facility at the National Institute of Oceanography, Haifa as described [15]. Large colonies were routinely subcloned into ramets of different sizes that were then tra