Figure 4.

TNF-α–induced MAPK but not NF-κB activation is dependent on FAK activity in vitro and in vivo. (A and B) FAK-WT or FAK-KD MEFs (A) and HUVECs pretreated with DMSO or FAK-I (1 µM PF271; B) were stimulated with 10 ng/ml TNF-α for the indicated times, and lysates were prepared for immunoblotting. Blots for activated FAK (pY397), total FAK, activated NF-κB (pS536), activated JNK (p-JNK and pT183/pY185), activated ERK (pERK and pT202/pY204), IκBα, and actin are shown. Internal loading controls for each gel are shown by anti-actin, anti-GAPDH, anti-talin, or reprobing membranes with antibodies to total NF-κB, JNK2, or total ERK1/2 immunoblotting. (C) PBS or TNF-α (0.02 mg/kg) was tail vein injected into mice, and, after 5 min (FAK and ERK activation) or 3 h (NF-κB activation), lung tissue was analyzed by immunoblotting or EMSA. Where indicated, FAK-I (100 mg/kg, PND-1186) was administered 3 h before starting experiments. Values, measured by densitometry, are means (±SD) from six mice, representing two independent experiments. ***, P < 0.001. (D and E) MEFs were replated onto FN dishes or held in suspension (Sus) for 1 h before TNF-α (10 ng/ml) addition for 15 min (D) or 6 h (E) before protein cell lysis. Blots for activated ERK (pERK and pT202/pY204), activated FAK (pY397), total FAK, and actin are shown. Anti-GAPDH blotting is shown as loading controls.