TNC-scTNF_R2 induces TNFR2 signaling in R2 MEF.

<p>(<b>A–C</b>) R2 MEF were stimulated with TNC-scTNF_R2 (20 ng/ml) for the indicated times. (<b>A</b>) TNFR2 was immunoprecipitated using MR2-1 antibodies and protein G agarose. The precipitates were analyzed by immunoblot analysis using anti-huTNFR2 (HP9003) and anti-TRAF2 antibodies (NB = non-bound; B = bound). (<b>B</b>) Localization of NFκB p65 was visualized via immunofluorescence microscopy as shown in the upper panel and the number of nuclei showing NFκB translocation was quantified. At least 200 cells per experiment were analyzed (n = 3; shown are the mean values ± SEM of percent NFκB positive nuclei; bar = 20 µm). (<b>C</b>) Phospho-Akt (Ser473) levels in cell lysates were analyzed using immunoblot analysis. Akt was used as a loading control. Representative blot and bar graph show the quantification of the phospho-Akt (Ser473) band. *p values less than 0.05 versus untreated cells were considered to be significant (n = 3; shown are the mean values ± SEM).