The pcDNA6/V5-TTP containing full-length ORF of human TTP [33 (link)] and the pGL3/TTPp-1343 containing human TTP promoter [29 (link)] were described previously. The pcDNA3-cMyc vector was purchased from Addgene.
For luciferase assays, cells were co-transfected with a pGL3/TTPp-1343-luciferase reporter construct and pRL-SV40 Renilla luciferase construct using TurboFectTM in vitro transfection reagent (Fermentas). Transfected cells were lysed with lysis buffer and mixed with luciferase assay reagent (Promega). The chemiluminescent signal was measured using a SpectraMax L Microplate (Molecular Devices, Sunnyvale, CA, USA). Firefly luciferase was normalized to Renilla luciferase in each sample. All luciferase assays reported in this study represent at least three independent experiments, each consisting of three wells per transfection.
Small interfering RNAs (siRNAs) against human TTP (TTP-siRNA, sc-36761), human c-Myc (c-Myc-siRNA, sc-29226), and control siRNA [scrambled siRNA (scRNA), sc-37007] were purchased from Santa Cruz Biotechnology (Santa Cruz). Cells were transfected 24 h after plating using LipofectamineTM RNAiMAX (Invitrogen) and were harvested at 48 h after transfection. The expression levels of TTP or c-Myc mRNA and protein were analyzed by RT-PCR and Western blotting, respectively.
For luciferase assays, cells were co-transfected with a pGL3/TTPp-1343-luciferase reporter construct and pRL-SV40 Renilla luciferase construct using TurboFectTM in vitro transfection reagent (Fermentas). Transfected cells were lysed with lysis buffer and mixed with luciferase assay reagent (Promega). The chemiluminescent signal was measured using a SpectraMax L Microplate (Molecular Devices, Sunnyvale, CA, USA). Firefly luciferase was normalized to Renilla luciferase in each sample. All luciferase assays reported in this study represent at least three independent experiments, each consisting of three wells per transfection.
Small interfering RNAs (siRNAs) against human TTP (TTP-siRNA, sc-36761), human c-Myc (c-Myc-siRNA, sc-29226), and control siRNA [scrambled siRNA (scRNA), sc-37007] were purchased from Santa Cruz Biotechnology (Santa Cruz). Cells were transfected 24 h after plating using LipofectamineTM RNAiMAX (Invitrogen) and were harvested at 48 h after transfection. The expression levels of TTP or c-Myc mRNA and protein were analyzed by RT-PCR and Western blotting, respectively.