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Mcherry rab5ca q79l

Manufactured by Addgene

MCherry-Rab5CA(Q79L) is a fluorescent protein construct that consists of the constitutively active mutant form of the Rab5 GTPase, Rab5CA(Q79L), fused to the mCherry red fluorescent protein. Rab5 is a small GTPase that regulates early endosome formation and trafficking. The Q79L mutation renders Rab5 constitutively active, leading to the formation of enlarged endosomes.

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4 protocols using mcherry rab5ca q79l

1

Fluorescent Rab Protein Constructs

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Wild-type monomeric red fluorescent protein (mRFP) Rab5 (pmRFP-C3) construct (Vonderheit and Helenius, 2005 (link)) was a gift from Ari Helenius (ETH Zurich; Addgene plasmid 14437). The constitutively active mCherry-Rab5CA(Q79L) (PmCherry-C1; Addgene plasmid 35138) and dominant-negative mCherry-Rab5DN(S34N) (PmCherry-C1; Addgene plasmid 35139) were gifts from Sergio Grinstein (University of Toronto; Bohdanowicz et al., 2012 (link)). The DsRed-Rab7 WT (pDsRed-C1; Addgene plasmid 12661), DsRed-Rab9 WT (pDsRed-C1; Addgene plasmid 12677), and DsRed-Rab11 WT (pDsRed-C1; Addgene plasmid 12679) were gifts from Richard Pagano (Mayo Clinic; Choudhury et al., 2002 (link)).
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2

Fluorescent Protein-Tagged Rab GTPases

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Nsg1-Emerald:Addgene cat#54202, Michael Davidson lab; the mutation at residue 114 from D to G was corrected.
Nsg1-mCherry:mouse full length Nsg1 was cloned at the N-terminal of mCherry in pcDNA3 vector.
Nsg2-GFP:mouse full length Nsg2 was cloned into pcDNA-GFP by Genscript
mRFP-Rab5:Addgene cat#14437, Ari Helenius lab.
mCherry Rab5CA(Q79L) cat#35138, mCherry Rab5DN(S34N) cat#35139:Addgene, Sergio Grinstein lab.
EGFP-Rab7 cat#12605, EGFP-Rab7DN(T22N) cat#12660:Addgene, Richard Pagano labmTagBFP2-Rab5, cat#56417, Addgene, Michael Davidson lab;
mTagBFP2-Rab7:Michael Davidson lab.
GFP:Clontech
mCherry:Clontech.
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3

Cloning and Tagging of Golgi Proteins

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Cloning of B4GT-EGFP-HT2 was previously described (Serebrenik et al., 2018 (link)). B4GT-SNAP-HT2 was cloned by replacing the EGFP domain of B4GT-EGFP-HT2 using Gibson assembly (Gibson et al., 2009 (link)). ST6GAL1-EGFP-HT2 and MAN2A1-EGFP-HT2 were cloned by replacing the transmembrane and cytoplasmic domain of B4GT-EGFP-HT2 with the first 113 and 88 amino acids of ST6GAL1 (Homo sapiens) and MAN2A1 (Mus musculus), respectively, via USER cloning (Nour-Eldin et al., 2010 ). B4GT-EGFP-DHFR* was created by replacing HT2 from B4GT-EGFP-HT2 with the DHFR domain from pBMN YFP-DHFR(DD) (a gift from Thomas Wandless [Stanford University], Addgene plasmid #29326). mCherry-Rab5CA(Q79L) was a gift from Sergio Grinstein (Addgene plasmid #35138). RFP-Rab5a and B4GT-GFP were gifts from James Rothman (Yale University), from which B4GT-RFP was derived by replacing the GFP domain with RFP via USER cloning. B4GT-3xFM*-GFP was a gift from Adam Linstedt (Carnegie Mellon University).
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4

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).
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