Expanded epithelial sheets from three explants cultured on plastic with DMSO treatment or on AM after treatment with DMSO, SB203580, or JNK inhibitor 1 for 17 days or treatment with LY294002, SR13668 or U0126 for 24 hours at day 10 (at this time point, cell outgrowth was rapid and there were enough cells to be collected for Western blot) were scraped off with a spatula. Cells were washed once with PBS, resuspended in 300 μL RIPA buffer (50 mM Tris Cl [pH 7.5]; 150 mM NaCl; 1% Nonidet P-40; 0.5% sodium deoxycholate; 0.1% SDS with 1 μg/mL of aprotinin, leupeptin, and pepstatin A; 1 mM phenylmethylsulfonyl fluoride [PMSF]; 50 mM sodium fluoride; and 0.2 mM sodium vanadate), and mixed at 4°C for 30 minutes. Cell lysates were cleared by centrifuging at 15,000g for 10 minutes at 4°C. The protein concentration in the supernatant was quantitated by bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL). Ten to 20 μg total protein from each sample was mixed with an equal volume of 2× SDS sample buffer, boiled for 5 minutes, electrophoresed on a 4% to 15% gradient SDS-PAGE gel and transferred to a nitrocellulose membrane. These membranes were preincubated with blocking buffer (5% BSA or nonfat milk in TBS/0.1% Tween-20), probed with an affinity-purified rabbit polyclonal (anti-Akt-pSer473, anti-FKHRL1-pThr32, anti-JNK MAPK, and anti-p44/42 MAPK pThr202/pTyr204), or mouse monoclonal antibody (anti-Akt-pThr308, anti-JNK-pThr183/Tyr185, anti-p38 MAPK, anti-p38 MAPK-pThr180/Tyr182, and anti-β-actin; all at 1:1000 dilution) overnight at 4°C. The membranes were then washed three times with TBS/0.1% Tween-20, incubated with goat anti-rabbit or rabbit anti-mouse IgG-peroxidase–conjugated antibody (Bio-Rad, Hercules, CA) for 1 hour at room temperature, and developed with enhanced chemiluminescence reagent (PerkinElmer Life Sciences, Boston, MA).