DLG2 (NM_001364) and DLG2 (NM_001351274.2) overexpression plasmids on a backbone of pcDNA3.1/C-(K)-DYK (OHu25658D and OHuq102626D respectively) vector were purchased from GenScript. LIN7A (NM_004664) over expression plasmid on a backbone of pCMV6-AC-GFP (PS100010) was purchased from Origene (RG221902). siRNA targeting DLG2 (s4122), LIN7A (s16836) or Silencer™ Select Negative control No. 1 siRNA (4390843) were purchased from Ambion (Thermo Fischer Scientific). SKNAS and HEK293 cells were grown to 80% confluence and subsequently transfected with; DYK-tagged DLG2 isoform 7, DYK-tagged DLG2 isoform 2, combined with GFP-tagged LIN7A, empty vector “mock” (pCMV6-Ac-GFP), si-LIN7A or scrambled negative control “mock”. 100 ng of DNA or 10 pmol siRNA was complexed with 0.3 µl of Lipofectamine 2000 according to the Lipofectamine 2000 reagent forward transfection protocol (Invitrogen; Thermo Fisher Scientific, Inc.).
Pcmv6 ac gfp ps100010
Manufactured by OriGene
Sourced in United States
PCMV6-AC-GFP (PS100010) is a plasmid vector designed for the expression of proteins fused with the green fluorescent protein (GFP) in mammalian cells. The vector contains the cytomegalovirus (CMV) promoter for high-level expression of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
1 sirna, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 reagent forward transfection protocol, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Mdig sirnas, by Qiagen (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
5 protocols using pcmv6 ac gfp ps100010
1
DLG2 and LIN7A Overexpression Plasmids
2
Modulating CIP2A and KRAS in Cells
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The siRNA construct targeting CIP2A (pLKO.1-shCIP2A, TRCN0000135532, target sequence: (5′-CCACAGTTTAAGTGGTGGAAA-3′) and non-targeting siRNA control (pLKO.1-shLuc) were obtained from the National RNAi Core Facility, Taiwan (http://rnai.genmed.sinica.edu.tw/index ). Lentivirus production and infection were performed as previously described [10 (link)]. The KRAS G12D mutant construct (pCMV6-Entry-KRAS G12D, RC400104) and control vector (pCMV6-AC-GFP, PS100010) were purchased from Origene (Rockville, Maryland, USA).
3
Knockdown and Overexpression of mdig in BEAS-2B Cells
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Control siRNA and mdig siRNAs were purchased from Qiagen (Valencia, CA). pCMV6-AC-GFP (PS100010) and pCMV6-AC-mdig-GFP (RG214829) were purchased from Origene (Rockville, MD). BEAS-2B cells, 3 × 105/well were seeded in 6-well plate, and grown to 70% confluency. siRNAs and plasmid DNA were transfected into cells using Lipofectamine RNAiMAX (Thermo Fisher Scientific) and Lipofectamine 2000 (Thermo Fisher Scientific), respectively, according to manufacturer's protocol. For rescue experiment, co-transfection of siRNA and mdig expression plasmid was performed using Lipofectamine 2000 according to manufacturer's protocol. Twenty four hours after transfection, cells were collected and subjected to Western blotting.
4
KRAS Mutant Expression in Cancer Cells
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PANC1 and MDA-MB-231 cells were transfected with plasmids containing the specified mutated open reading frame (ORF) clones (no 3′ UTR) of KRAS. The KRAS G12D mutant construct (pCMV6-Entry-KRAS G12D, RC400104), KRAS G13D mutant construct (pCMV6-Entry-KRAS G12D, RC400116), and control vector (pCMV6-AC-GFP, PS100010) were purchased from OriGene (Rockville, MD, USA). KRAS protein expression was verified by western blotting.
5
Transcriptional Regulation by KLF14 in HEK293
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Human embryonic kidney (HEK) 293 cells were purchased from the American Type Culture Collection (ATCC). All cell culture products were purchased from Invitrogen Gibco. HEK293 cells were cultured in a 5% CO 2 humidi ed incubator at 37 °C. Cells (∼60% con uence)
were transfected using jet PRIMETM (Polyplus-transfection, USA) for 48 h. The mouse KLF14 eukaryotic expression vector pCMV6-AC-GFP-KLF14 (MG221172) and the KLF14 empty vector pCMV6-AC-GFP (PS100010) were purchased from Origene (Origene, USA). For the reporter assays, the cells were cultured in a 24-well plate and then co-transfected with the dual luciferase reporter plasmid pGL3-Basic-Btg2 promoter or pGL3-Basic, pCMV6-AC-GFP-KLF14 or pCMV6-AC-GFP and the renilla luciferase reporter gene plasmid (pGMLR-TK luciferase reporter plasmid). Fire y and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (E1910, Promega, USA). Renilla luciferase activity was normalized to re y luciferase activity. Each experiment was repeated at least three times.
were transfected using jet PRIMETM (Polyplus-transfection, USA) for 48 h. The mouse KLF14 eukaryotic expression vector pCMV6-AC-GFP-KLF14 (MG221172) and the KLF14 empty vector pCMV6-AC-GFP (PS100010) were purchased from Origene (Origene, USA). For the reporter assays, the cells were cultured in a 24-well plate and then co-transfected with the dual luciferase reporter plasmid pGL3-Basic-Btg2 promoter or pGL3-Basic, pCMV6-AC-GFP-KLF14 or pCMV6-AC-GFP and the renilla luciferase reporter gene plasmid (pGMLR-TK luciferase reporter plasmid). Fire y and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (E1910, Promega, USA). Renilla luciferase activity was normalized to re y luciferase activity. Each experiment was repeated at least three times.
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