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. 2014 Feb 7;3(2):252–264. doi: 10.1002/cam4.198

Figure 2.

Figure 2

HIF-1α, REDD1, mTOR downstream proteins and pAMPK in PC-3M cells exposed to MSeA in presence of wortmannin in hypoxia. PC-3M cells were pretreated for 1 h with 1 μmol/L wortmannin followed by 6 h treatment with MSeA in the absence or presence of wortmannin. (A) Nuclear extracts were processed for HIF-1α and Lamin B xpression levels and cytosolic fractions were analyzed for REDD1, native AKT, pAKT, pRPS6, 4E-BP1, pAMPK and β-actin protein levels. *Overexposed Western blot for visualization of pRPS6 in the presence of wortmannin. (B) Fold changes for target proteins estimated from immunoblots in (A) compared to control untreated PC-3M cells using ImageJ analysis. The fold change in band densities of HIF-1α were normalized to the band densities of the respective Lamin B levels of the nuclear extracts in all samples. The fold change in band densities of pAKT was normalized to the band densities of the respective native AKT and to β-actin levels. While for REDD1, pRPS6, 4E-BP1, and pAMPK the band densities were normalized to β-actin levels. HIF-1α, hypoxia-inducible factor-1 alpha; REDD1, regulated in development and DNA damage 1; MSeA, methylseleninic acid.