Megfp c1

Manufactured by Addgene

MEGFP-C1 is a plasmid that encodes a monomeric enhanced green fluorescent protein (mEGFP) for expression in mammalian cells. The plasmid contains a cytomegalovirus (CMV) promoter to drive the expression of mEGFP.

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Lab products found in correlation

Megfp n1, by Addgene (2 mentions) Pmig pp2aa, by Addgene (2 mentions) Pmig pp2ab, by Addgene (2 mentions) Pcdna3.1 drp1, by Addgene (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Memerald sec61b c1, by Addgene (1 mentions) Er mrfp, by Addgene (1 mentions) Opti mem, by Thermo Fisher Scientific (1 mentions) Pmcherry c1, by Takara Bio (1 mentions)

6 protocols using megfp c1

Fluorescent Protein Plasmid Construction

Cited 3 times

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ER-mRFP (Addgene no. 62236), mTagRFP-T2-Mito-7 (Addgene no. 58041) (referred to as mitoRFP in the text), mTagBFP2-N1 (Addgene no. 54566), mEGFP-N1 (Addgene no. 54767), mEGFP-C1 (Addgene no. 54759) and mEmerald-Sec61b-C1 (Addgene no. 90992) have been described previously, and were gifts from Erik Snapp, Michael Davidson, or generated in house. EGFP-VAPB^{59 (link)}, HA-PTPIP51 (ref. ^{16 (link)}) and pHAGE-Tet-STEMCCA^{60 (link)} have been previously described and were gifts from Pietro De Camilli, Kurt De Vos and Robert Tijan, respectively.
All insertions and cassette changes were performed using the NEBuilder implementation of Gibson Assembly (New England Biolabs) unless specified otherwise, taking care to leave appropriate restriction sites for later changes. All constructs were sequenced before use and will be available on Addgene unless prohibited by copyright. Specific strategies and resulting plasmid maps are linked in the Supplementary Information.

Obara C.J., Nixon-Abell J., Moore A.S., Riccio F., Hoffman D.P., Shtengel G., Xu C.S., Schaefer K., Pasolli H.A., Masson J.B., Hess H.F., Calderon C.P., Blackstone C, & Lippincott-Schwartz J. (2024). Motion of VAPB molecules reveals ER–mitochondria contact site subdomains. Nature, 626(7997), 169-176.

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Phosphomimetic FUS Variants in Cellular Assays

Cited 1 time

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Wild-type FUS and its phosphomimetic variants were subcloned from pET vectors previously constructed (Monahan et al., 2017 (link)) into both mEGFP-C1 (Addgene 54759) and pIRES2-EGFP-SHP2DA (Addgene 12286) using BamHI/XhoI restriction sites. The phosphomimetic substitution in each construct are listed below. Plasmid DNA was transfected into cell lines using OptiMEM (Life Technologies 31985-070) transfection medium and Lipofectamine 2000 (Thermo Fisher 11668027) at a ratio of 0.5 µg: 2.5 µl (DNA: Lipofectamine 2000). Transfection mixture was diluted 1:4 into complete cell media and incubated at 37°C for 8–10 h.
Phosphomimetic substitutions in FUS are diagrammed in Supplemental Figure S8 and are as follows: FUS-1E (T19E); FUS-3E (S26E, S30E, T68E); FUS-4E (T68E, S84E, S87E, S117E); FUS-6E (S26E, S30E, T68E, S84E, S87E, S117E); FUS-12E (T7E, T11E, T19E, S26E, S30E, S42E, S61E, T68E, S84E, S87E, S117E S131E).

Rhoads S.N., Monahan Z.T., Yee D.S., Leung A.Y., Newcombe C.G., O’Meally R.N., Cole R.N, & Shewmaker F.P. (2018). The prionlike domain of FUS is multiphosphorylated following DNA damage without altering nuclear localization. Molecular Biology of the Cell, 29(15), 1786-1797.

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Cloning and Expression of G3BP Paralogs

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All G3BP paralogs (G3BP1, G3BP2A, and G3BP2B) cDNAs were cloned by Gibson assembly (NEB, E2611L) into either an mEGFP-C1 (Addgene, 54759) or mEGFP-N1 (Addgene, 54767) plasmid with Kanamycin resistance cassettes. Fusion proteins were interspaced with a glycine-serine (GS) linker of 8 residues to improve folding and stability of flanking domains. G3BP1/2 mutants were generated by site directed mutagenesis. For lentivirus expression, these synthetic constructs were cloned by Gibson assembly into a lentivirus backbone with a SFFV promoter, and an ampicillin resistance cassette for bacterial selection. For the generation of cell lines with labeled nuclei, a H2B-mCherry construct cloned in a lentivirus backbone was kindly shared by Dr. Xiaokun Shu (University of California, San Francisco, USA). For in vitro reconstitution, NTF2 domain of G3BP1 variants was cloned into a pET28 MBP-TEV bacterial backbone (Addgene, 69929) with Gibson assembly.

Liboy-Lugo J.M., Espinoza C.A., Sheu-Gruttadauria J., Park J.E., Xu A., Jowhar Z., Gao A.L., Carmona-Negrón J.A., Wittmann T., Jura N, & Floor S.N. (2024). Protein-protein interactions with G3BPs drive stress granule condensation and gene expression changes under cellular stress. bioRxiv.

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Molecular Cloning of Fluorescent Fusion Proteins

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pmCherry-C1 (632524, Clontech) was used as the backbone and control vector for the overexpression experiments. mCherry–ARHGEF7 was a gift from Dorus Gadella (Addgene #129611). The cDNA of RASGRF2 was cloned into pmCherry-C1 from R777-E241 Hs.RASGRF2, a gift from Dominic Esposito (Addgene #70525). mEGFP-C1 was a gift from M. Davidson (Addgene #54759). pLKO-RFP-shCntrl, a gift from William Kaelin (Addgene #69040), was used as the backbone and control vector for the knockdown experiments. Sequences of short hairpin RNAs (shRNAs) against ARHGAP23 or SRGAP1 can be found in Supplementary Table 12a.

Wang L., Pang K., Zhou L., Cebrián-Silla A., González-Granero S., Wang S., Bi Q., White M.L., Ho B., Li J., Li T., Perez Y., Huang E.J., Winkler E.A., Paredes M.F., Kovner R., Sestan N., Pollen A.A., Liu P., Li J., Piao X., García-Verdugo J.M., Alvarez-Buylla A., Liu Z, & Kriegstein A.R. (2023). A cross-species proteomic map reveals neoteny of human synapse development. Nature, 622(7981), 112-119.

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Plasmid Cloning and Lentiviral Production

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pMIG-PP2Aa (#10884), pMIG-PP2Ab (#13804) and pcDNA3.1-Drp1 (#34706) plasmids were purchased from Addgene and subcloned into mEGFP-C1 (#54759) and pCMV-Myc-3B vectors. RalA^WT, RalA^G23V and RalA^S28N plasmids were subcloned into a pLVX vector with 3× Flag tag for lentiviral production.

Xia W., Veeragandham P., Cao Y., Xu Y., Rhyne T.E., Qian J., Hung C.W., Zhao P., Jones Y., Gao H., Liddle C., Yu R.T., Downes M., Evans R.M., Rydén M., Wabitsch M., Wang Z., Hakozaki H., Schöneberg J., Reilly S.M., Huang J, & Saltiel A.R. (2024). Obesity causes mitochondrial fragmentation and dysfunction in white adipocytes due to RalA activation. Nature Metabolism, 6(2), 273-289.

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Molecular Cloning of RalA Variants

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pMIG-PP2Aa (#10884), pMIG-PP2Ab (#13804), and pcDNA3.1-Drp1 (#34706) plasmids were purchased from Addgene and subcloned into mEGFP-C1 (#54759) and pCMV-Myc-3B vectors. RalA^WT, RalA^G23V, and RalA^S28N plasmids were subcloned into a pLVX vector with 3x Flag tag for lentiviral production.

xia W., Veeragandham P., Cao Y., Xu Y., Rhyne T., Qian J., Hung C.W., Zhao P., Jones Y., Gao H., Liddle C., Yu R., Downes M., Evans R., Ryden M., Wabitsch M., Reilly S., Huang J, & Saltiel A. (2023). Obesity-dependent increase in RalA activity disrupts mitochondrial dynamics in white adipocytes. Research Square.

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