Effect of GILZ on TNF-α-induced ERK phosphorylation and Smurf expression.

<p>(A, B) MSC-GFP and MSC-GILZ cells were serum starved for 48 hr and then treated with TNF-α (1 ng/ml) for 15 min before harvesting. Equal amounts of total cell lysates were separated on SDS PAGE gel, transferred onto membrane and analyzed for levels of phosphorylated ERK1/2 (pERK). Levels of pERK were quantified with NIH Image J software and presented as a ratio of pERK/total ERK in B. Value from untreated MSC-GFP is arbitrarily set as 1. Experiments were repeated three times with similar results. Levels of overexpressed GILZ are also shown. Error bars indicate SD. α-tubulin, instead of β-actin, was used as a loading control in these experiments because β-actin and ERK have similar molecular weights. (C–F) RNA and protein samples from MSC-GFP and MSC-GILZ cells used in the experiments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031717#pone-0031717-g002" target="_blank">figure 2</a> were analyzed by real-time qRT-PCR and Western blot to show the levels of Smurf1 and Smurf 2 mRNA (C, D) and protein (E). Quantitative results of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031717#pone-0031717-g003" target="_blank">Fig. 3E</a> are shown as bar graphs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031717#pone-0031717-g003" target="_blank">Fig. 3F and G</a>, respectively. Data were normalized to β-actin and expressed as fold changes relative to control cells cultured in GM. These experiments were repeated three times with similar results. Error bars indicate SD. a, <i>p</i><0.01 (n = 3).