TM cells were serum starved for 24 hours and exposed to various agents as indicated. Lysates were harvested in RIPA lysis buffer (20 mM Tris-HCl [pH 7.4], 50 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 5 mM NaF, 1 mM PMSF, 1 mM Na3VO4, 1 μM leupeptin, and 0.3 μM aprotinin). The lysates were clarified by centrifugation at 14,000 rpm for 20 minutes and the samples were normalized for total protein. Protein concentration in the cell lysates was determined with a Bradford Assay (Bio-Rad, Hemel Hempstead, UK). For Western blot analysis, 10 μg of TM protein extract was loaded onto each lane and separated by SDS-PAGE. The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Watford, UK), blocked with 5% (wt/vol) nonfat dried milk in TBST for 1 hour at room temperature, and probed overnight at 4°C with antibodies raised against β-actin (1:10,000;Sigma-Aldrich), phospho p42/44 (1:1,000), total p42/44 (1:1,000), phospho p38 (1:1,000), total p38 (1:1,000), and phospho JNK (1:1,000) all Cell Signaling Technology, Danvers, MA). Membranes were incubated with HRP-conjugated secondary Abs (1:2000) for 1 hour at room temperature, and proteins were visualized by chemiluminescence. Unless otherwise stated, the antibodies used were supplied by Cell Signaling Technology. Images of developed photographic film were acquired with a CCD camera and captured with Image-acquisition and -analysis software (Visionworks LS; UVP, Upland, CA). Densitometric analysis of band intensity was performed (Image, ver. 4.0; Scion Corp., Frederick MD).