Expression profile of MSK1 and p-MSK1 (Thr-581 and Ser-360) following LPS intracerebral injection.

<p><b>A</b>. Protein levels of t-MSK1, p-MSK1 Thr-581, p-MSK1 Ser-360 were detected before (control) and after injury. GAPDH was also detected by Western blotting. <b>B</b>. Quantification graphs (relative optical density) of the intensity of staining of p-MSK1 (Thr-581) and total MSK1 to GAPDH at each time point. GAPDH was used to confirm that equal amounts of protein were run on the gel. <b>C–H</b>. Immunofluorescence staining of MSK1 and p-MSk1 (Thr581) was performed to assess the staining changes for MSK1 and p-MSK1 immunoreactivity in the cortex at day 1 after LPS-injection. <b>I</b>. Negative control. * and ^# indicate significant differences at P<0.05, compared with normal brain cortex. Scale bars: 40 µm (C–F), 20 µm (G–J).</p

Immunolocalization of MSK1 and p-MSK1 (Thr-581) with different cellular markers in cerebral cortex by double immunofluorescence staining.

<p>In the adult rat brain cortex, within 5(red, <b>A</b> and <b>E</b>) and p-MSK1 (Thr581) (red, <b>I</b> and <b>M</b>) and different cell markers (green, <b>B, F, J, N</b>), such as a neuronal marker (NeuN) and an astrocyte marker (GFAP). The yellow color in the merged images represents colocalization of MSK1 or p-MSK1 (Thr581) with different phenotype-specific markers (<b>C, G, K, O</b>). Colocalization of MSK1 and p-MSK1 (Thr581) with different phenotype-specific markers in the normal group are shown in the brain cortex (<b>D, H, L, P</b>). Quantitative analysis of different phenotype-specific marker-positive cells expressing MSK1 (<b>Q</b>) and p-MSK1 (<b>R</b>) (%) in the unit area (mm²) in the normal group and 1 day after injury. *indicates significant difference at P<0.05, compared with the normal group. Error bars indicate SEM. Scale bars: 20 µm (<b>A–P</b>).</p

Effects of MSK1 gene silencing and Thr-581 mutation on cytokine production in LPS-treated astrocytes.

<p><b>A</b>. Effects of siRNA for MSK1 and non-specific siRNA on LPS-induced expression of MSK1, p-MSK1 (Thr581) and iNOS were detected by Western blotting. <b>B</b>. The bar chart shows the ratio of total MSK1 and p-MSK1 to β-actin. <b>C–E</b>. ELISA showed that MSK1 gene silencing by siRNA further promoted the LPS-mediated upregulation of inflammatory cytokines. <b>F</b>. Western blot analysis showed the effect of mutation of Thr-581 to an alanine residue on LPS-induced expression of p-MSK1 Thr-581, p-MSK1 Ser-360, and total MSK1. <b>G</b>. The bar chart shows the ratio of p-MSK1 Thr-581 and p-MSK1 Ser-360 to total MSK1. <b>H–J</b>. ELISA showed the effect of mutation of Thr-581 on LPS-induced TNFα, IL-6, and IL1-β production in activated astrocytes.</p