FIG. 3.

LPS activation of MEK in RAW264.7 cells is dependent on TPL-2 and proteasome activity. (A) RAW 264.7 cells stably transfected with expression vectors encoding wild-type Myc-TPL-2 (WT), kinase-inactive Myc-TPL-2 (KD), or no insert control (empty vector [EV]) were stimulated with LPS, and cell lysates were subjected to Western blotting. LPS activation of endogenous MEK and p38 activation were assayed using phospho-specific antibodies. (B to D) RAW264.7 cells were preincubated with MG132 (40 μM) or DMSO vehicle control for 30 min and then stimulated with LPS for the times indicated. (B) Total cell lysates were subjected to Western blotting for the indicated proteins. LPS activation of MEK and p38 activation were monitored with the phospho-specific antibodies. (C) Cell lysates were immunodepleted of p105 and Western blotted for TPL-2 and α-tubulin. (D) TPL-2 was immunoprecipitated from total cell lysates and then assayed for MEK kinase activity as for Fig. 1A.