Analysis of the differential effect of n-Butanol, U0126, Wortmannin and LY294002 on reporter gene expression and phosphorylation of mTOR in transfected PC3 and 293T cells.

<p>Effect of n-butanol on expression of (<b>A</b>) GFP; (<b>B</b>) β-Gal, and (<b>C</b>) Inhibition of phosphorylation of mTOR by n-butanol; Effect of U0126 (U0), Wortmannin (Wort) and LY294002 (LY) on (<b>D</b>) β-Gal and (<b>E</b>) GFP expression. Proteins were separated by 12–14% SDS-PAGE and detected by immunoblotting using antibodies specific to GFP and β-Gal.</p

Comparative analysis of the translational regulatory proteins and different signaling pathway proteins in PC3 and 293 T cells.

<p>(<b>A</b>) Analysis of mTOR target proteins 4EBP1, S6, eEF2K and p70S6K. (<b>B</b>) mTOR and TSC2. (<b>C</b>) PI3K/Akt and ERK1/2. (<b>D</b>) PKC. 200 µg of cell lysate was used for PKC analysis and 50 µg was used for analysis of other proteins.</p

Analysis of RNA and protein levels derived from the transfected expression vectors of PDGF-B, GFP and β-Gal in PC3, PC3BM, HeLa, 293T and MA104 cells.

<p>(<b>A</b>) PDGF-B mRNA levels were determined by RNase protection Assay. The 144 nt protected band corresponds to PDGF-B and the 120 nt band represents that of β-Actin mRNA. (<b>B</b>) RT-PCR of β-Gal, GFP and β-Actin mRNA in pcDNA3-β-Gal and pcDNA3-GFP transfected cells. (<b>C</b>) Radioimmunoprecipitation of PDGF-B protein expressed in pCMV-PDGF-B transfected cells using an N-terminal antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014408#pone.0014408-Rao2" target="_blank">[69]</a>. (<b>D</b>) Levels of β-Gal, GFP and β-Tubulin proteins in pcDNA3- β-Gal and -GFP transfected cells. 50 µg of transfected cell lysate was analyzed for GFP and β-Gal levels by SDS-PAGE. (<b>E</b>) β-galactosidase assay using the β-Gal ELISA Kit from Roche Diagnostics. (<b>F</b>) Fluorescence microscopy and bright field (BF) images of 293T, PC3BM, HeLa and PC3 cells transfected with pcDNA-GFP reporter gene construct. (<b>G</b>) Analysis of the fold differences in expression of the reporter mRNA and protein levels between PC3 and HeLa, and PC3 and PC3BM.</p

Western blot analysis of key target proteins of mTOR pathway.

<p>Analysis of total protein and/or phosphorylated forms of (<b>A</b>) mTOR and TSC2, (<b>B</b>) eIF4E, (<b>C</b>) 4EBP1, (<b>D</b>) S6, (<b>E</b>) eEF2 and eEF2K (<b>F</b>) p70S6K, (<b>G</b>) PI3K and PDK1, (<b>H</b>) Akt and (<b>I</b>) ERK1/2, p38 MAPK and PKC. 50 µg of cell lysate was used for analysis of mTOR, TSC2, PI3K, PDK1, Akt, eEF2, 4EBP1 and S6 and 100 µg was used for detection of phosphorylated forms and other proteins.</p