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. 2012 Mar 23;7(3):e32989. doi: 10.1371/journal.pone.0032989

Figure 4. UTRs are not required for PEDF downregulation by hypoxia in SBcl2 melanoma.

Figure 4

Western blot analysis from SBcl2-pCEP4 and SBcl2-pCEP4-PEDF melanoma cell lines incubated with 1 mM DMOG or under hypoxia (1% O2): (A) extracellular PEDF (PEDFe) and Penta-HIS (5-HIS) protein levels in 24 h conditioned medium (CM) and (B) intracellular PEDF (PEDFi) and HIF1α protein levels in whole-cells extract. β-tubulin was used as loading control. (C) Western blot analysis of PEDFe protein levels in 24 h CM, PEDFi and HIF1α protein levels in whole-cell extracts from SBcl2 melanoma cell line incubated under normoxia (21% O2) or hypoxia (1% O2). β-tubulin was used as loading control. (D) UTR-reporter assay in SBcl2 melanoma cell line transfected with a Renilla promoter reporter containing the 3′ UTR of PEDF (psiCHECK2-3′PEDF), the 3′UTR of GAPDH (psiCHECK2-3′GAPDH) or an empty reporter (psiCHECK2) and incubated under normoxia or hypoxia for 24 h. Renilla activity was normalized to luciferase activity, which is used as an internal control of transfection efficiency. psiCHECK2-3′GAPDH was used as a negative control. Bars represent average ± standard deviation (SD).