pIRES, pIRES-SIK1, pIRES-SIK1K56M, pIRES-SIK1S577A and pEGFPC-SIK1 were a kind gift from Dr. Hiroshi Takemori, National Institute of Biomedical Innovation, Japan. The plasmids pEF5/FRT/V5/GW-CAT, pEF5/FRT/ICER I and pEF5/FRT/ICER IIγ were constructed as previously described [20] . Empty control-vector was constructed by restriction cutting with BsrGI in the att sites of pEF5/FRT/V5-DEST. Gastrin-17 and cycloheximide were purchased from Sigma Chemical (St. Louis, MO). pSRE-luc and pc-fos-luc were a kind gift from Prof. Ugo Moens, University of Tromsø, Norway [21] (link). pCRE-luc containing four CRE somatostatin consensus promoter elements (TGACGTCA) and pNF-κB-luc, containing five copies of the NF-κB enhancer element (TGGGGACTTTCCGC ), were obtained from Stratagene (La Jolla, CA). pAP1-luc containing four tandem copies of the AP1 consensus sequence (TGA(G/C)TCA) was obtained from Clontech (Palo Alto, CA). HDAC5 Flag plasmid was obtained from Add Gene Plasmid (# 13822).
Pap1 luc
Manufactured by Takara Bio
Sourced in Japan, United States
The PAP1-luc is a lab equipment product from Takara Bio. It is a plasmid construct that contains the luciferase reporter gene under the control of the PAP1 promoter.
Automatically generated - may contain errors
Lab products found in correlation
Pnf κb luc, by Takara Bio (5 mentions)
Phrl tk renilla luciferase reporter, by Promega (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (2 mentions)
Dual luciferase assay kit, by Promega (2 mentions)
Pcre luc, by Takara Bio (2 mentions)
Psre luc, by Takara Bio (2 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Control sirna, by Horizon Discovery (1 mentions)
Phrl tk plasmid, by Promega (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Anti β actin a1978, by Merck Group (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Anti ha, by Roche (1 mentions)
Anti phospho creb, by Cell Signaling Technology (1 mentions)
Fudeltagw rtta, by Addgene (1 mentions)
Fuw teto hoct4, by Addgene (1 mentions)
Prl tk, by Promega (1 mentions)
Pmscv puro retroviral vector, by Takara Bio (1 mentions)
12 protocols using pap1 luc
1
Plasmid Constructs for SIK1 Signaling Studies
2
Tip110 and USP15 Plasmids and Knockdown
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Tip110.His, Tip110ΔN.His, Tip110ΔC.His, GFP-Tip110ΔNLS, Tip110ΔNLS.His, and GFP-Tip110 plasmids were described elsewhere (28 (link), 74 (link)). Tip110Δ274-963.His, Tip110Δ387-963.His, Tip110Δ557-963.His, and Tip110Δ786-963.His plasmids were constructed in the backbone of pcDNA3 (Invitrogen) using standard PCR techniques with Tip110.His as a template and using EcoRI and XhoI cloning sites. USP15.Myc (1–952 aa), USP15ΔC.Myc (1–384 aa) and USP15ΔN.Myc (385-952 aa) were described elsewhere (25 (link)). NF-κB p65 was a kindly provided by Dr. Michael Klemsz of Indiana University School of Medicine. pNF-κB-luc and pAP-1-Luc were purchased from Clontech Laboratories Inc., CA. UB.HA plasmid was kindly provided by Dr. Mark Hannink of the University of Missouri. IκBα.HA plasmid was kindly provided by Dr. Michael Karin of the University of California (75 (link)). The IκBα-Nuc.Myc plasmid was constructed using IκBα.HA as a template, backbone of pCMV-Nuc-Myc (Addgene) and using SalI and NotI cloning sites. On-TARGETplus Tip110 siRNA (L-013447-01) and SiRNA control (D-001810-01) were purchased from Dharmacon.
3
Quantifying NF-κB and AP-1 Transcriptional Activity
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The pathway reporter of pNFκB-Luc and pAP-1-Luc plasmids, which containing NF-κB or AP-1 binding sites in the promoter region, were purchased from Clontech (Mountain View). Overnight cultured RAW264.7 cells in 96-well plates were transfected with 100 ng of pNFκB-Luc or pAP-1-Luc reporter vector and 5 ng of phRL-TK Renilla luciferase reporter (Promega) using Lipofectamine™ 2000 transfection reagent (Invitrogen). 16 h after transfection, cells were treated with various concentrations of berry extracts and LPS/IFN-γ (for NF-κB reporter) or PMA (for AP-1 reporter), simultaneously. After another 24 h, the cells were lysed for Firefly/Renilla luciferase activity assay using a dual-luciferase assay kit (Promega). The relative luciferase activities were normalized by LPS/IFN-γ or PMA control.
4
Macrophage NF-κB and AP-1 Luciferase Assay
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RAW 264.7 macrophages were cotransfected with pNF-κB-luc or pAP-1-luc (Clontech, Shiga, Japan) plasmid plus the phRL-TK plasmid (Promega, WI, USA) using Lipofectamine LTX™ (Invitrogen, CA, USA) as instructed by the manufacturers. After 24 h of transfections, cells were pretreated with KCT-01 for 1 h prior to LPS (1 μg/mL) stimulation. After 18 h, each well was washed with cold-PBS and cells were lysed and the luciferase activity was determined using the Promega luciferase assay system (WI, USA).
5
Functional Regulation of Nm23 Tumor Suppressors
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The cDNA of RGS19 was purchased from Missouri S&T cDNA Resource Center (Rolla, MO, USA). The cDNAs of human Nm23H1 and Nm23H2 were kindly provided by Dr. Patricia S. Steeg (National Cancer Institute) and Dr. Tina H. Lee (Carnegie Mellon University), respectively. Luciferase reporter genes pCRE-luc, pSRE-luc and pSTAT3-luc were from Clontech (Palo Alto, CA), and pAP-1-luc was kindly provided by Prof. Karl Tsim (Division of Life Science, Hong Kong University of Science and Technology). HEK293, MDA-MB-231, MDA-MB-435s, A549, and HeLa cells were purchased from and authenticated by the American Type Culture Collection (Rockville, MD, USA). Cells were cultured in accordance to ATCC recommendations. Transfections were performed using LipofectAMINE 2000 reagent (Life Technologies, Grand Island, NY) according to the manufacturer’s protocol. For stable transfection in MDA-MB-231 cells, cells were selected by 0.5 mg/mL G418 for 2-3 weeks. Expression of Nm23H1/2 was confirmed by immunoblots. Anti-β-actin (A1978) was from Sigma-Aldrich, anti-HA (11666606001) from Roche, anti-CREB (#9197), anti-phospho-CREB (#9198) and anti-Nm23H1/2 (#3338) were from Cell Signaling Technology.
6
Regulatory Transcription Factor Plasmid Library
7
Cloning and Profiling of miR-424-5p
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The human miR-424-5p gene was PCR amplified from genomic DNA and cloned into a pMSCV-puro retroviral vector (Clontech, Japan). Pathway Profiling System, including pAP1-luc, pCRE-luc, pE2F-TA-luc, pERE-luc, pGRE-luc, pHSE-luc, pISRE-TA-luc, pMYC-luc, pNF-κB-TA-luc, pp53-TA-luc, pSRE-luc, and pSTAT3-TA-luc, was purchased from Clontech (PT3286-1). HOPFlash (Catalog [Cat.] 83467), HIPFlash (Cat. 83466), TOPFlash (Cat. 12456), and FOPFlash (Cat. 12457) were purchased from Addgene. The 3′ UTRs of WWC1, SAV1, and LATS2 were PCR amplified from genomic DNA and cloned into pmirGLO vectors (Promega, USA), and the list of primers used in cloning reactions was provided in Table S3 . Anti-miR-424-5p was synthesized and purified by GENECHEM (China). The primers of the mutant plasmid for WWC1, SAV1, and LAST2 were synthesized and purified by HDbio (Guangzhou, China). Transfection of plasmids was performed as previously described.46 (link)
8
Pol ι Overexpression Luciferase Assay
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Pol ι overexpression and knockdown cells grown in 24-cell plates were transfected with 1 μg of pAP1-Luc (Clontech, USA) vector for 36h. Cell lysates were prepared using the lysis buffer contained in the Dual-Luciferase reporter assay kit (Promega, USA) and luciferase activity was measured by a luminometer (Promega-GloMax, USA). Relative luciferase activity was normalized by co-transfection with 50 ng pRL-SV40 vector and analysis of Ranilla luciferase activity.
9
Transcriptional Regulation Assay in HEK293 and MDA-MB-231 Cells
10
Transcriptional Regulation by NF-κB and AP-1
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The pathway reporter plasmids of phosphorylated (p)NF-κB-Luc and phosphorylated activated protein 1 (pAP-1)-Luc, which contain NF-κB or AP-1 binding sites in the promoter region, were purchased from Clontech Laboratories, Inc. Overnight cultured RAW264.7 cells in 96-well plates were transfected with 100 ng pNFκB-Luc or pAP-1-Luc reporter vector and 5 ng phRL-TK Renilla luciferase reporter (Promega Corporation) using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). At 16 h post-transfection, the cells were treated with various concentrations of BM and LPS (for NF-κB reporter). After another 24 h, the cells were lysed for Firefly/Renilla luciferase activity assay using a dual-luciferase assay kit (Promega Corporation). The relative luciferase activities were normalized by LPS/IFN-c or PMA control.
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