SP600125 prevents Cx43 ubiquitination in LPS-treated rat astrocytes.

<p>(<b>A</b>) Astrocytes were treated with 2 µg/ml of LPS for 0, 3, 6, 8, or 10 h. The cell lysates immunopreciptated using normal rabbit serum (<b>NRS,</b> immunoprecipitation control, <b>IPC</b>) or rabbit antibody against total Cx43 (<b>IP: Cx43</b>). The immunoprecipitates were then subjected to 10% SDS-PAGE and immunoblotting using antibodies against ubiquitin (<b>IB: Ubi</b>). The immunoprecipitates were then subjected to 10% SDS-PAGE and immunoblotting using antibodies against ubiquitin (<b>IB: Ubi</b>) or total Cx43 (<b>IB: Cx43</b>). (<b>B</b>) The blots from 3 independent experiments were subjected to densitometric analyses for ubiquitinated Cx43 and the results expressed as the density of the bands in the test sample relative to those in the time zero sample. *<i>P</i><0.05, NS (not significant) compared to 0 h using Dunnett’s post-hoc test. (<b>C</b>) The cell lysates from astrocytes treated with 0.05% DMSO (<b>Cont</b>), 2 µg/ml of LPS (<b>LPS</b>) for 6 h, or were pretreated for 30 min with 10 µM SP600125 followed by LPS (<b>LPS+SP</b>) treatment for 6 h in the presence of the inhibitor were immunopreciptated using normal rabbit serum (<b>NRS,</b> immunoprecipitation control, <b>IPC</b>) or rabbit antibody against total Cx43 (<b>IP: Cx43</b>). The immunoprecipitates were then subjected to 10% SDS-PAGE and immunoblotting using antibodies against ubiquitin (<b>IB: Ubi</b>), Nedd4 (<b>IB: Nedd4</b>) or total Cx43 (<b>IB: Cx43</b>). (<b>D</b>) The blots from 3 independent experiments were subjected to densitometric analyses for Nedd4 or ubiquitinated Cx43 and the results expressed as the density of the bands in the test sample relative to those in the control. *<i>P</i><0.05, **<i>P</i><0.01 compared to the LPS-treated group using Dunnett’s post-hoc test.