CDKN1B 3′ UTR was subcloned into psiCHECK-2 vector (Promega, USA) using XhoI and PmeI restriction enzymes (New England Biolabs, USA). Wild-type PBE sequences 5′-TGTATATA-3′ was mutant to 5′-acaATATA-3′ as previous report[12 (link)]. Cells were co-transfected with pCMV6-hPUM1 and luciferase reporter plasmids, containing fragments or full-length 3′ UTRs. After 48 hours, cells were washed with PBS and lysed in Passive Lysis Buffer (Chroma-Glo Luciferase Assay System, Promega). Then, 20 μL of each lysate was analyzed using the Dual-Luciferase Reporter Assay System (Chroma-Glo Luciferase Assay System, Promega) in a 96 Microplate luminometer (BioTek, USA).
Protocol detail
PUM1 regulation of CDKN1B 3' UTR
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Corresponding Organization :
Other organizations : Nanjing Medical University, Second Affiliated Hospital of Nanjing Medical University, Northwestern University
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Variable analysis
independent variables
- PCMV6-hPUM1
- Luciferase reporter activity
- PsiCHECK-2 vector
- Wild-type PBE sequences
- Mutant PBE sequences (5'-acaATATA-3')
- PBS for cell washing
- Passive Lysis Buffer
- Dual-Luciferase Reporter Assay System
- 96 Microplate luminometer
- Positive control: Not specified
- Negative control: Not specified
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