TPL2 is required for PsaDM-dependent ERK1/ERK2 MAPK phosphorylation in AECs. A.

<p>BEAS-2B AECs were transfected with increasing amounts of siRNA (0 to 32 µM) directed against TPL2 as indicated. Following lysis, 30 µg of lysates was submitted to SDS-PAGE followed by immunoblotting with an antibody that recognizes all form of TPL2. <b>B.</b> BEAS-2B AECs were transfected with control or TPL2 siRNA (32 µM) for 72 hours then left untreated or exposed to PsaDM for 15 minutes. ERK1/ERK2 was immunoblotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059116#pone-0059116-g001" target="_blank"><b>Fig. 1</b></a>. <b>C–D.</b> BEAS-2B AECs were pre-treated 1 hour with increasing doses of the TPL2 inhibitor C1 (0–3 µM) and exposed to 5 µg/ml PsaDM for 15 minutes. ERK1/ERK2 phosphorylation was determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059116#pone-0059116-g001" target="_blank"><b>Fig. 1</b></a> (<b>C</b>), while p38 phosphorylation was evaluated by immunoblotting with antibodies recognizing only the phosphorylated forms of p38α or antibodies that recognize all forms of p38α (<b>D</b>). <b>E.</b> BEAS-2B AECs were left untreated or pre-treated for 1 hour with vehicle, 2 µM C1 or 2 µM PD184352 (a MKK1/MKK2 inhibitor) and stimulated with 50 ng/mL EGF for 30 minutes. EGFR and ERK1/ERK2 were immunoblotted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059116#pone-0059116-g001" target="_blank"><b>Fig. 1</b></a>. Representative blots from four distinct experiments are shown (left panels). Quantitative analysis of the signals was performed and expressed as graphs (right panels).