Pcdna4 vps34 flag

Manufactured by Addgene

Sourced in United States

The pcDNA4-VPS34-FLAG is a plasmid vector that expresses the VPS34 protein with a FLAG tag. VPS34 is a phosphoinositide 3-kinase that plays a role in the regulation of various cellular processes, including autophagy and endocytosis. The FLAG tag allows for the detection and purification of the expressed VPS34 protein.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Atg16l1, by OriGene (1 mentions) Human msr1, by OriGene (1 mentions) Prk5 ha ubiquitin plasmid, by Addgene (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Sartorius (1 mentions) Hek293, by National Collection of Authenticated Cell Cultures (1 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions) Ptag bfp c h rab5a c myc, by Addgene (1 mentions) Mcherry rab5ca q79l, by Addgene (1 mentions) Kod plus mutagenesis kit, by Toyobo (1 mentions)

6 protocols using pcdna4 vps34 flag

Molecular Cloning of Autophagy Proteins

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The Myc-DDK-tagged mouse Msr1 ORF clone (Cat# MR205384), Atg12 (Cat# MR200886), Atg5 (Cat# MR203691), Atg16l1 (Cat# MR209513), Atg9a (Cat# MR208753), Atg13 (Cat# MR207683) and, p62/Sqstm1 (Cat# MR226105), human MSR1 (Cat# RC209609) and Beclin 1 (Cat# MR207162) were obtained from Origene (Rockville, MD 20850, USA). pcDNA-FLAG-Ulk1 (Plasmid # 27636)^{62 (link)} and pcDNA4-VPS34-FLAG (Plasmid # 24398)^{63 (link)} were purchased from Addgene (Cambridge, MA 02139, USA). Myc-Msr1 was constructed by inserting Msr1 ORF (amplified from Origene clone MR205384) into the pcDNA3.0-Myc vector. The LR2006-OPY1 CHIKV genes were amplified by PCR and subcloned into pcDNA3.0-Myc. Human Atg16l1, Atg16l1ΔFBD (amino residues 229–242 deleted) and Atg16l1ΔWD40 (amino residues 337-end) amplified by PCR and inserted into pcDNA3.1 (Zeo)-FLAG vector. The primers are listed in Supplementary Table 1.

Yang L., Geng T., Yang G., Ma J., Wang L., Ketkar H., Yang D., Lin T., Hwang J., Zhu S., Wang Y., Dai J., You F., Cheng G., Vella A.T., Flavell R.A., Fikrig E, & Wang P. (2020). Macrophage scavenger receptor 1 controls Chikungunya virus infection through autophagy in mice. Communications Biology, 3, 556.

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Ubiquitin Mutant Transfection in HEK293 and HT22 Cells

Cited 1 time

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HEK293 (human embryonic kidney) and HT22 (mouse hippocampal neuron) cell lines were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's modified eagle medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Biological Industries). Both cell lines were cultured at 37 °C in a humidified 5% CO ₂ atm.
pcDNA4-Vps34-Flag was purchased from Addgene (#24398). All ubiquitin mutants were made from pRK5-HA-ubiquitin plasmid (Addgene) by mutagenesis. pRK5-HA-Ubiquitin-K0 (no lysine), pRK5-HA-Ubiquitin-K6 (keep lysine at position 6), pRK5-HA-Ubiquitin-K11, pRK5-HA-Ubiquitin-K27, pRK5-HA-Ubiquitin-K29, pRK5-HA-Ubiquitin-K33, pRK5-HA-Ubiquitin-K48, pRK5-HA-Ubiquitin-K63. 24 h before transfection, cells were seeded to obtain a final confluence of 60–70%. Transfections were carried out using Lipofectamine™ 2000 (Thermo Fisher Scientific, #11668019) according to manufacturer's protocols. PcDNA3.1 was used as an expression control. siRNA sequence for UBE2N was: GAACCAGTTCCTGGCATCA (RiboBio silencer, ID#stB000527A).¹⁵ (link)

Tang J., Sun F., Deng X.J., Ma Y.L., Li K.Y., Tang Y, & Chen G.J. (2019). Ubiquitination status does not affect Vps34 degradation. Genes & Diseases, 7(3), 401-407.

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Vps34 Regulation by Umbelliprenin

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Following culture in the 6 cm plates for 24 h, BxPC3 cells were transiently transfected with 4 μg of pCDNA4‐Vps34‐Flag (#24398, addgene) plasmid with Lipofectamine 3000 (Invitrogen) and then treated with umbelliprenin or DMSO. The cell lysates containing 500 μg of total protein were mixed with 1 μg of Vps34 antibody and protein A/G beads overnight. The samples were separated by SDS‐PAGE and visualized with enhanced ECL reagents.

Wang H., Liu Y., Wang Y., Xu T., Xia G, & Huang X. (2023). Umbelliprenin induces autophagy and apoptosis while inhibits cancer cell stemness in pancreatic cancer cells. Cancer Medicine, 12(14), 15277-15288.

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BCRP3 Regulation via shRNA

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BCRP3 cDNA was amplified from the genomic DNA of BT474 cells using the primers: Fw: 5′ CCGGAATTCACTCCGTAGTGTGCACTTGGT and Rv: 5′ACGCGTCGACTTTTCGTCTCAGGAAACTTTTTAATG. The cDNA was subcloned to the lentiviral vector pLAS5w.Pneo. BCRP3 shRNAs were predicted via BLOCK-iT™ RNAi Designer (https://rnaidesigner.thermofisher.com/), generated by Purigo Biotechnology, Taipei, Taiwan, and cloned to pLKO.1. The shRNA targeting sequences are as follows: shLuc 5′TTACGCTGAGTACTTCGA; shBCRP3#1 5′ATATTGGACGCTGGACCCGC; shBCRP3#2 5′ GACGCTGGACCCGCAGGCCC. Plasmids encoding EGFP-2xFYVE, HA-Beclin 1, and FLAG-Beclin 1 were kindly provided by Guang-Chao Chen (Academia Sinica, Taipei, Taiwan). Plasmids encoding GFP-DFCP1, GFP-LC3, and RFP-p62 were kindly provided by Wei Yuan Yang (Academia Sinica). pcDNA4-VPS34-FLAG was purchased from Addgene, Watertown, MA, USA (#24398). The 4 × SBE-Luc construct was obtained from Rik Derynck (University of California at San Francisco).

Yan R.L., Luan C.L., Liao C.C., Liu L.H., Chen F.Y., Chen H.Y, & Chen R.H. (2022). Long noncoding RNA BCRP3 stimulates VPS34 and autophagy activities to promote protein homeostasis and cell survival. Journal of Biomedical Science, 29, 30.

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Autophagy Regulation by TcdB Glucosyltransferase

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The GFP-LC3 construct was kindly provided by Dr. Li Yu (Tsinghua University). pCMV-myc-ATG7 (24921) and pcDNA4-VPS34-Flag (24398) were purchased from Addgene. pEF-ATG14L, pEF-UVRAG were constructed onto pEF6-BSD-myc/his-B vector. pHis1522-TcdB plasmid was kindly provided by Dr. Hanping Feng (University of Maryland). pHis1522-TcdB-mutant (Y284A &D286N/D288N) was cloned by site mutagenesis. pEF6-BSD-B was used to construct plasmids expressing either glucosyltransferase domain of TcdB or its mutant form (Y284A &D286/288 N). pCMV5-3xFlag-ATG7 was constructed into pCMV5 vector. Primers and other information are listed in supplementary materials.

He R., Peng J., Yuan P., Yang J., Wu X., Wang Y, & Wei W. (2017). Glucosyltransferase Activity of Clostridium difficile Toxin B Triggers Autophagy-mediated Cell Growth Arrest. Scientific Reports, 7, 10532.

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Rab5-Mediated Vesicle Dynamics Protocol

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pET-GST-R5BD-hRABEP1 was purchased from Vector Builder. Rab5a-pmCherryC1 was a gift from Christien Merrifield (Addgene plasmid #27679; http://n2t.net/addgene:27679, accessed on 31 May 2019; RRID:Addgene_27679) [41 (link)]. pcDNA4-Vps34-Flag was from Qing Zhong (Addgene plasmid #24398; http://n2t.net/addgene:24398, accessed on 12 November 2018; RRID:Addgene_24398) [42 (link)]. mCherry-Rab5CA(Q79L) was from Sergio Grinstein (Addgene plasmid #35138; http://n2t.net/addgene:35138, accessed on 26 April 2018; RRID:Addgene_35138) [43 (link)]. TagRFP-T-EEA1 was from Silvia Corvera (Addgene plasmid #42635; http://n2t.net/addgene:42635, accessed on 9 November 2017; RRID:Addgene_42635) [44 (link)]. pTag-BFP-C-h-Rab5a-c-Myc was from James Johnson (Addgene plasmid #79801; http://n2t.net/addgene:79801, accessed on 9 November 2017; RRID:Addgene_79801) [45 (link)]. RFP-Rab5 140 and 180 mutants were generated with primers (5′-GCAGCAGCTGTTGACTTCCAGG-3′ and 5′-ATTTGCTAAGTCAGCTTTGTTTCCTGAC-3′) and (5′-GCAGCGCTGCCAAAGAATGAAC-3′ and 5′-AGCTATTGCCATAAATATTTCATTTACATTCATTG-3′), respectively, using a KOD-plus mutagenesis kit (Toyobo, Osaka, Japan).

Mashimo M., Morozumi A., Nobeyama A., Kanzaki M., Negi S., Kato J., Moss J., Nomura A, & Fujii T. (2022). Poly(ADP-ribose) Polymerase 1 Mediates Rab5 Inactivation after DNA Damage. International Journal of Molecular Sciences, 23(14), 7827.

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