1 h at room temperature. Covers were then incubated with mouse monoclonal antibody Body against b tubulin in blocking buffer for 1 h at room temperature or overnight at 4 C diluted incubated After washing three times in PBS with 0.05% Tween 20, cells were incubated Roscovitine Seliciclib with FITC -conjugated goat anti-mouse IgG for 45 min at room temperature. The cells were again as described above and then observed under a confocal laser beam washed. Statistical analysis The data were pr as mean standard deviation Presents. Student, St-test was used to compare the difference between the two groups. The difference was considered statistically significant if p \ 0.05. Results nocodazole, the activation of p38 in response to extracellular Re stimuli Rat 1 cells are rat and fibroblasts were used successfully to the regulation of p38 activation in response to extracellular To study re stimuli.
Therefore, the M Possibility was that the destruction Tion of microtubules k Nnte the AZD1152-HQPA Aurora Kinase inhibitor activation of p38 in response to extracellular Re inhibit stimuli in rat-1 cells. For this purpose, rats-1 cells were incubated with 1 lg / ml nocodazole or DMSO treated the same volume for 4 h and then a Immunfluoreszenzf Staining. The data in FIG. 1a shows that in fact destroy nocodazole Rt long fadenf Shaped structure of the microtubules as expected. Microtubules are required to run the cell cycle. Were subjected to determine whether the same treatment leads to cell cycle arrest, the cells to cell cycle analysis. As shown in Fig. 1b, 1 lg / ml nocodazole for 4 h resulted in a slight increase in G2 / M Bev Lkerung.
Thus, the aberrant Kinaseaktivit t not due to differences in cell cycle distribution. To investigate the regulation of p38 by microtubules, rat-1 cells were pretreated with VX-745 nocodazole or DMSO of equal volume, followed by stimulation with or without 20 J/m2 UV and then min Final incubation for 30 min. Immunoblot analysis showed that phosphorylation of UV-induced p38 at Thr180 and Tyr182, which is required for the activation of p38, was significantly inhibited by nocodazole. Thus, our data point out in rat-1 cells, nocodazole also inhibits the activation of p38 in response to extracellular Re stimuli. Nocodazole, the activation of p38 in response to extracellular Re stimuli in a transcription dependent Ngig, but p38 activity Independent t Way ngiger We then tested whether nocodazole yourself k Nnte the activation of p38 in the cells of the rat .
induce For this purpose, rats-1 cells were incubated with 1 lg / ml nocodazole or DMSO treated the same volume for 4 h and then subjected to immunoblotting. The data in FIG. 2a showed that nocodazole activated by p38 itself weakly. Nocodazole to test whether the activation of p38 in response to extracellular Re stimuli via a negative feedback mechanism inhibited, a rat, cells were pretreated with or without nocodazole, followed by treatment with TNF in the presence or absence of the inhibitor of RNA synthesis actinomycin D or selective inhibitor of p38 SB203580. This treatment protocol has been shown that blockade of transcription and the functional inhibition of p38 kinase activity T 1 in rat cells Immunoblotting showed that TNF inhibition by nocodazole-induced p38 activation by actinomycin D but was not abolished by SB203580.