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Pwzl neo myr flag pkm2

Manufactured by Addgene

The PWZL Neo Myr Flag PKM2 is a laboratory equipment product. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.

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2 protocols using pwzl neo myr flag pkm2

1

Plasmid Construction and Cloning Protocol

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The vectors pWZL Neo Myr Flag PKM2 and pEGFP-C1-PKM2 were purchased from Addgene (plasmid #20585 and plasmid #64698, deposited by Jean Zhao and Axel Ullrich, respectively). The Glut1 promoter reporter clone (pEZXPG02.1-Glut1) was purchased from Genecopoeia (USA). The pTIG1-myc vector has been described previously (Wu et al., 2011 (link)). The DNAJC8 cDNA fragment was amplified from pDNAJC8/PACT2 using 5′ (5′-TGGCTAGCATGGCGGCTTCA GGAGAGAGC-3′) and 3′ (5′-GACTCGAGCTCACGTTGCTCC ATTTTTACTTTCGG-3′) primers and then was subcloned inframe into the NheI-XhoI sites of PCR3.1-Flag (Dr. Yi-Ling Lin, Institute of Biomedical Science, Academia Sinica, Taiwan) to generate pDNAJC8-Flag. The DNAJC8 cDNA fragment was amplified from pDNAJC8-Flag using 5′ (5′-TCGAATTCTATGG CGGCTTCAGGAG-3′) and 3′ (5′-GTGGATCCCTCACGTTGCT CCATTTTTA-3′) primers and then subcloned in-frame into the EcoRI-BamHI sites of the pEGFP-C1 vector (Clontech Laboratories, USA) to generate pEGFP-DNAJC8. The PKM2 cDNA fragment was amplified from pWZL Neo Myr Flag PKM2 using 5′ (5′-TCGAATTCGATGTCGAAGCCCCATAGTG-3′) and 3′ (5′-GTGGATCC CGGCACAGGAACAACACGC-3′) primers and then subcloned in-frame into the EcoRI-BamHI sites of the pcDNA-myc-His vector (Invitrogen, USA) to generate pPKM2-myc.
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2

Transient Transfection of Plasmids in Cells

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Plasmids, including pWZL Neo Myr Flag PKM2 and pMXs-hcMYC were obtained from Addgene (Cambridge, MA). pcDNA Flag GFP (Flag-GFP) was preserved in our laboratory and was used as control. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, CA) according to the manufacturer’s instructions. Briefly, PC-3 or MCF-7 cells were plated onto a 96-well plate. When the cells are cultured to approximately 85% confluence, the media were replaced with OPTI-MEM. Then, 3 µg plasmids and 30 µl lipofectamine reagent was mixed in a tube containing 1500 µl OPTI-MEM by vigorous vortexing. After incubation for 15 min, the mixture was added to the cell cultures and incubated for certain times.
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