Herrmann und Ulrike / Band 2 (German Edition)

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Articles

  1. Kindle Feature Spotlight
  2. Implicit Critique
  3. Genotype-Independent Transmission of Transgenic Fluorophore Protein by Boar Spermatozoa
  4. 3.09 Hermann Cohen: Germanness and Judaism (Germany, 1916)
  5. Genotype-Independent Transmission of Transgenic Fluorophore Protein by Boar Spermatozoa

For quantitative FRAP analysis the overall setup remained largely unchanged. Bleaching was performed with 25 iterations after 2 seconds of initial data recording. Cell lysates were centrifuged at 18, g for 10 minutes. Western blot analysis was performed with the indicated antibodies and the enhanced chemiluminescence kit Amersham, Arlington Heights, USA according to the manufacturer's instructions.

Bromouridine incorporation was performed according to the protocol by Hendzel et al. Briefly, cells were pre-incubated with 1 mM bromouridine Sigma-Aldrich, Germany for 1 hour before fixation. Immunostaining was performed using the antibodies as indicated, i. Antibodies were diluted 1: Cells were incubated for 45 minutes with the specific first antibody raised against the protein of interest and then with the secondary antibody conjugated with TRITC Dako, Hamburg, Germany.

All stainings were performed at room temperature. Domain borders were chosen according to Becker et al.

Kindle Feature Spotlight

After stripping, the blot was reprobed with a STAT3 antibody lower panel. Mean values of experiments performed in triplicate are depicted. Moreover, the occurence of this band does not depend on stimulation. STAT3 appears heterogeneously distributed within the nucleus, with the exclusion of the nucleolus.

In many stimulated cells small dot-like structures were observed. These dots were not visible in unstimulated cells. When cells were transfected with STAT3, the dot-like structures appeared with increased intensity but again only in stimulated cells Fig.

Implicit Critique

STAT3 in nuclear bodies. Images were taken by confocal microscopy. Endogenous upper panels as well as transfected lower panels STAT3 was detected. Images taken at the timepoints indicated are depicted. A time-lapse movie can be downloaded as supplementary data on the homepage of the Journal of Cell Science jcs. After stripping the blots were reprobed with a STAT3 antibody for loading control. After these initial observations, we were interested in whether the formation of these structures could be studied by live cell imaging using STAT3-YFP.

Genotype-Independent Transmission of Transgenic Fluorophore Protein by Boar Spermatozoa

STAT3 appears in nuclear bodies between 10 and 13 minutes after stimulation. The fluorescence intensity of the nuclear bodies increases until about 25 minutes of stimulation. Subsequently, the intensity decreases until the nuclear bodies disappear after about minutes. In unstimulated cells STAT3 phosphotyrosine staining resulted in only a weak background signal. On stimulation, the amount of tyrosine-phosphorylated STAT3 increases throughout the cell.

3.09 Hermann Cohen: Germanness and Judaism (Germany, 1916)

Phosphorylation of STAT3 in nuclear bodies. Cells were analyzed by confocal microscopy. Similarly, phosphorylation of STAT3 at serine was analyzed. In unstimulated cells STAT3 serine phosphorylation was not detected in the cytosol or in the nucleus Fig. Interestingly, on stimulation serine-phosphorylated STAT3-YFP was detected within the nucleus but hardly in the cytosol, indicating that serine-phosphorylated STAT3 is present predominantly in the nucleus of stimulated cells Fig.

Thus, STAT3 in nuclear bodies is phosphorylated at tyrosine and serine Subsequently, recovery of the fluorescence was observed by live cell imaging Fig. Recovery of fluorescence at the location of the original dot was observed within a few seconds after bleaching. Subsequently, the intensity of the recovered dot remained constant but did not reach the intensity of the dot before bleaching.

Subsequently, images were taken at the timepoints indicated. Fluorescence before bleaching was normalized to YFP was transfected into HepG2 cells as a control for a freely mobile protein. Unstimulated cells were analyzed as described above black graph. Each graph represents the mean values of five independent experiments.

Great German Aces of WWII

As a reference for a freely diffusing protein, cells expressing YFP alone were also analyzed. Small circular regions of interest ROI were bleached so that recovery of fluorescence could be continuously monitored immediately after bleaching. The graphs in Fig. The rapid increases in fluorescence intensities immediately after the bleaches are characteristic of freely diffusing proteins. The values of the mobile fractions of YFP After photobleaching, STAT3-YFP fluorescence within nuclear bodies also recovers rapidly but reaches only half of the initial value Thus, STAT3 within nuclear bodies consists of a highly mobile and an immobile fraction.

From the experiments presented so far it can be concluded that activated STAT3 concentrates within nuclear bodies. Within this structure the mobility of STAT3 is restricted, but still a rapid exchange with the nuclear environment occurs. Therefore, we performed costainings of STAT3 with proteins resident in these subnuclear structures, i. Cells were stimulated with IL-6 for 30 minutes or left unstimulated as indictated. Cells were stimulated with IL-6 for 30 minutes or left unstimulated. Previous studies have shown that post-translational modification of proteins by sumoylation can lead to a change in subnuclear localization of the modified protein Chakrabarti et al.

Immunofluorescence and western blot analysis failed to detect any sumoylation of STAT3 data not shown. Therefore, we conclude that STAT3 in nuclear bodies is not a primary target of sumoylation. STAT3 was identified as a transcription factor that induces acute phase protein genes in response to IL-6 Wegenka et al.

Therefore, one would expect that nuclear STAT3 is localized at sites of active gene transcription. As expected, CBP Fig. Similar observations were made when nascent RNA was labeled by bromouridine incorporation. The bromouridine staining shows newly synthesized RNA concentrated in diffuse dot-like structures. These results suggest that STAT3 nuclear bodies are associated with sites of active transcription.

Fortyeight hours after transfection cells were pre-incubated in medium supplemented with 1 mM bromouridine for 30 minutes. Images of fixed cells were taken by confocal laser-scanning microscopy upper panel. Intensities of the BrU and STAT3-YFP signals are also shown in false-color mode, indicating highest intensities in white and red and lowest intensities in blue and black lower panel.

Is active transcription required for the formation of STAT3 nuclear bodies? Thus, although STAT3 nuclear bodies are associated with transcriptionally active chromatin, transcription itself is not required for STAT3 body formation. Images of fixed cells were taken by confocal laser-scanning microscopy. DMSO treatment alone was taken as a control.

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Genotype-Independent Transmission of Transgenic Fluorophore Protein by Boar Spermatozoa

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