Fig. 2. The TPL2 pathway regulates TNF production mainly at the post-translational level. (A) BMDM from wild-type (wt, black squares) and TPL2-deficient (–/–, white squares) mice were either left untreated or were stimulated with 10 ng/ml LPS for the times (hours) indicated. The levels of TNF in the cell culture supernatant were analysed by standard ELISA. Each square represents a determination using a different mouse. (B) BMDM were lysed in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1 mM Na₃VO₄, 100 nM okadaic acid, 2 mM Na₄P₂O₇, a mixture of protease inhibitors and 1% Nonidet-P 40. Lysates from wild-type and TPL2-deficient mice were either left untreated or were stimulated with 100 ng/ml LPS for the times (minutes) indicated, and were immunoblotted for pre-TNF as in Fig. 1 or for tubulin. (C) The experiment was carried out as in A except that total RNA was purified from the lysates, reverse transcribed and the amount determined by real-time quantitative PCR.