TRAF2 expression is downregulated in PDAC cell lines.

<p><b>A:</b> Cell lysates of indicated cells were normalized to 0.5 mg/ml and then 20 µg were subjected to SDS-PAGE. Samples were transferred to nitrocellulose and analyzed by Western blot for expression of TRAF2 (anti-TRAF2), TRAF3 (anti-TRAF3), cIAP1 (anti-cIAP1), cIAP2 (anti-cIAP2) or β-actin (anti-β-actin; loading control). TRAF2 overexpressed in Hek293 cells served as an additional molecular weight control. <b>B:</b> Samples of mRNA of indicated cell lines were subjected to quantitative RT-PCR directed against TRAF2. Samples were normalized to GAPDH. <b>C:</b> Panc1 cells (5×10⁵ cells, 6 cm dishes) were co-transfected with TRAF2 and vector control or HA-ubiquitin. After 24 hours cells were lysed, TRAF2 immunoprecipitated (anti-TRAF2) and analyzed by immunoblotting for ubiquitination of TRAF2 (anti-HA). Blots were re-probed for TRAF2 (anti-TRAF2). <b>D:</b> Indicated cell lines were treated with MG-132 (20 µM) for 0, 4, 8, 16 or 24 hours. Cells were lysed and analyzed for expression of endogenous TRAF2 (anti-TRAF2) or β-actin (anti-β-actin; loading control. TRAF2 overexpressed in Hek293 cells served as positive control. <b>E:</b> Panc1 cells (5×10⁵ cells, 6 cm dishes) were transfected with vector control or TRAF2 as indicated. After 24 hours cell lysates were analyzed by Western blotting for expression of NIK (anti-NIK), overexpressed TRAF2 (anti-TRAF2) or β-actin (anti-β-actin) as loading control.