Journal: Oncotarget
Article Title: Inhibition of PHLPP2/cyclin D1 protein translation contributes to the tumor suppressive effect of NFκB2 (p100)
doi: 10.18632/oncotarget.8746
Figure Lengend Snippet: ( A – C ) The cell extracts as indicated were subjected to Western Blot and β-Actin was used as a protein loading control (top panel). Total RNA was isolated and was then subjected to RT-PCR analysis of larp7 mRNA expression in the indicated cells and β-actin mRNA was used as a loading control (bottom panel). ( D and E ) The larp7 promoter-driven luciferase reporter was transiently transfected into the cells as indicated and luciferase activity of each transfectant was evaluated upon seeded in 96-well plate for 48 hrs. The results were presented as relative larp7 promoter activity and the symbol (*) indicates a significant difference of larp7 promoter activities between two groups ( p < 0.05). ( F ) Schematic representation of the transcription factors binding sites in the mouse larp7 promoter-driven luciferase reporter. ( G – I ) The cell extracts as indicated were subjected to Western Blot and β-Actin was used as a protein loading control. ( J ) Schematic representation of CREB point mutant of the larp7/miR302d promoter-driven luciferase reporter. ( K ) Wild-type larp7/miR-302d promoter-driven luciferase reporter or its mutant at CREB binding site, were co-transfected with pRL-TK into p100+/+ and p100−/− cells. Luciferase activity of each transfectant was evaluated and the results were presented as relative larp7 promoter activity. The symbol (*) indicates a significant difference ( p < 0.05). ( L ) The cell extracts as indicated were subjected to Western Blot and β-Actin was used as a protein loading control. ( M ) The miR-302d expression in the indicated cells was determined by real-time PCR and the symbol (*) indicates a significant inhibition of miR-302d expression as compared with vector transfectant ( p < 0.05). ( N ) cyclin d1 3′-UTR luciferase reporter was transiently transfected into p100−/−(p100/Nonsense) and p100−/−(p100/shCREB) cells. Luciferase activity of each transfectant was evaluated and the results were presented as relative cyclin d1 3′-UTR activity. The symbol (*) indicates a significant difference of cyclin d1 3′-UTR activities in comparison to p100−/−(Nonsense) cells ( p < 0.05).
Article Snippet: The shRNA constructs specific targeting mouse and human p100 and mouse CREB were purchased from Open Biosystems (Thermo Fisher Scientific, Pittsburgh, PA).
Techniques: Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Luciferase, Transfection, Activity Assay, Binding Assay, Mutagenesis, Real-time Polymerase Chain Reaction, Inhibition, Plasmid Preparation
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