For transient transfection of hippocampal neurons, 300 ng of plasmid DNA were transfected using 1 μl of Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) in 100 μl of the plating medium. Neurons were transfected at DIV 8 and were fixed after two additional days of incubation. Plasmids used were as follows: pEGFP-N1 (Takara Bio, Mountain View, CA, USA), mCherry-Rab5CA (Q79L; Addgene, #35138), mCherry-Rab5DN (S34N; Addgene, #35139), mRFP-Rab5 (Addgene, #14437), GFP-rab7 WT (Addgene, #12605), GFP-rab7 DN (Addgene, #12660), EGFP-Rab7A Q67L (Addgene, #28049), EGFP-Rab4A (Addgene, #49434), EGFP-Rab4AQ67L (Addgene, #49475), EGFP-Rab4AS22N (Addgene, #49476), HA-Rab11-DN (S25N), (Addgene, #101046), HA-Rab11-WT (Addgene, #101047), EGFP-Rab11AQ70L (Addgene, #49553), HA-Rab8a-WT (Addgene, #101048), HA-Rab8a-DN (T22N; Addgene, #101049), and HA-Rab8a-CA (Q67L) (Addgene, #101050). Inhibitors were dissolved in dimethylsulfoxide (DMSO), HGF (Merck KGaA, Darmstadt, Germany) in PBS and added to hippocampal neurons (DIV 9) to final concentrations of 50 ng/ml for HGF, 200 nm for mTOR inhibitor rapamycin (Merck), and 1 μM PHA-665752 (Merck KGaA, Darmstadt, Germany). Analyses were performed 30 min and 24 h (HGF Stimulation) or 24 h (Rapamycin treatment) after the addition of the reagents.
Mrfp rab5
Manufactured by Addgene
Sourced in United States
MRFP-Rab5 is a fluorescent protein fusion construct that consists of the monomeric red fluorescent protein (mRFP) fused to the Rab5 small GTPase. Rab5 is a key regulator of early endosome dynamics and trafficking. The mRFP-Rab5 fusion protein enables visualization of early endosome localization and trafficking in live cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Mda mb 231ln, by PerkinElmer (2 mentions)
Human timp2 cdna, by Thermo Fisher Scientific (2 mentions)
Hs578t, by American Type Culture Collection (2 mentions)
Hek293t, by American Type Culture Collection (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Mcherry rab5ca, by Addgene (1 mentions)
Mcherry rab5dn, by Addgene (1 mentions)
Egfp rab4aq67l, by Addgene (1 mentions)
Egfp rab4as22n, by Addgene (1 mentions)
Egfp rab11aq70l, by Addgene (1 mentions)
Mtor inhibitor rapamycin, by Merck Group (1 mentions)
Hepatocyte growth factor (hgf), by Merck Group (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Gfp rab7 wt, by Addgene (1 mentions)
Egfp rab7a q67l, by Addgene (1 mentions)
Egfp rab4a, by Addgene (1 mentions)
Pha 665752, by Merck Group (1 mentions)
Mrfp rab7, by Addgene (1 mentions)
7 protocols using mrfp rab5
1
Transient Transfection of Hippocampal Neurons
2
Molecular Tools for Cell Line Analysis
3
Plasmid Cloning for Autophagy Study
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All primer sequences used in these experiments are listed in Supplementary Table 1 . MT, 2xMT, CA, CA-MT, and CA(C258S) were generated by PCR amplification of pcDNA3.1(−)-Flag-RavZ and pcDNA3.1(−)-Flag-RavZ(C258S) vectors and inserted into the N3-GFP or C1-GFP vectors using each restriction enzyme set. CA(Δα3)-MT was amplified by overlap extension PCR with C1-CFP-CA-MT used as a template, and then was inserted into the C1-GFP vector using the restriction enzyme set. GFP-AKT1-PH, GFP-Lact-C2, mRFP-Rab5 and mRFP-Rab7 were provided by Addgene (Cambridge, MA, USA). We used previously described DNA constructs for mRFP-LC3B, and mRFP-GABA RAP in this study.
4
Visualizing Endocytic Pathways with Fluorescent Fusion Proteins
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pmRFP-Rab5 was obtained from Addgene (Addgene plasmid 14437). pcDNA4-EGFP-DTX1 and pcDNA4-LAMP1-mCherry were generated as described previously9 (link). To generate pcDNA4-cerulean-c-FLIPL, cerulean fragments were amplified by PCR from Cerulean-GalT (Addgene plasmid 11930) with HindIII/EcoRI and cloned into pcDNA4-c-FLIPL-Myc with HindIII/EcoRI to create a translational fusion construct.
293T cells (1 × 106) transduced with mRFP-Rab5 or LAMP1-mCherry were transfected with cerulean-c-FLIPL and EGFP-DTX1. 24 h after transfection, cells were re-seeded on a 22 × 22 mm glass coverslip and allowed to attach for another 18 h. Cells were fixed in 2% paraformaldehyde in PBS for 15 min at 37 °C and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. Cells were mounted in Dapi-Fluoromount-GTM (SouthernBiotech, Birmingham, AL) and observed under a Zeiss LSM 780 confocal microscope.
293T cells (1 × 106) transduced with mRFP-Rab5 or LAMP1-mCherry were transfected with cerulean-c-FLIPL and EGFP-DTX1. 24 h after transfection, cells were re-seeded on a 22 × 22 mm glass coverslip and allowed to attach for another 18 h. Cells were fixed in 2% paraformaldehyde in PBS for 15 min at 37 °C and permeabilized with 0.3% Triton X-100 for 10 min at room temperature. Cells were mounted in Dapi-Fluoromount-GTM (SouthernBiotech, Birmingham, AL) and observed under a Zeiss LSM 780 confocal microscope.
5
Optimized APPL1 and EEA1 Colocalization
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Wild-type HeLa cells were transfected with pEGFPC1-human APPL1, a gift from Pietro De Camilli (Addgene plasmid #22198)6 (link); EEA1 TagRFP-T, a gift from Silvia Corvera (Addgene plasmid #42635)24 (link); EGFP-EEA1, a gift from Silvia Corvera (Addgene plasmid #42307)13 (link); EGFP-Rab5, a gift from Marci Scidmore (Addgene plasmid #49888); and mRFP-Rab5, a gift from Ari Helenius (Addgene plasmid #14437)69 (link). Cells were transfected with a total of 1 µg DNA (0.3 µg + 0.3 µg plasmid of interest + 0.4 µg blank DNA) using lipofectamine 3000 (Thermo Fisher Scientific). The DNA sequence for the N-terminal mutant of EEA1 carrying F41A and I42A, deficient in Rab5 binding, was synthesised and cloned into the TagRFP-T vector using the XhoI/BamHI sites. For the C-terminal binding mutant carrying R1375A, the synthesised sequence was cloned into the TagRFP-T vector using the XhoI/BamHI sites. It has been reported that drastic over-expression of APPL1 or EEA1 results in colocalisation of APPL1 and EEA1 on Rab5 endosomes; we, therefore, optimised this concentration by screening for this artefact and choosing conditions where we observed no overlap of APPL1 and EEA1.
6
Molecular Tools for Cell Line Analysis
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Cell lines HEK293T (ATCC), Hs578T (ATCC), and MDA-MB-231LN (Caliper LifeScience) were grown based on the manufacturer's recommendations. shRNAs and siRNAs against NEDD9, non-targeting control (ThermoFisher Scientific), and NEDD9-cDNA were prepared as previously described (7 (link)). mRFP-Rab5, and -Rab7 were purchased from Addgene (plasmid #14437, #14436) (60 (link)). Plasmids mCherry-MMP14 and PAmCherry-MMP14 were a gift from Dr.Chavrier (Institute Curie, France) and Dr.Machesky (The Beatson Institute for Cancer Research, Scotland) (24 (link)). Human TIMP2-cDNA (ThermoFisher Scientific) was sub-cloned into pcDNA3.1-3x-Flag plasmid (gift from Dr.Frisch, West Virginia University, USA).
7
Cloning and Characterization of KIF5B Constructs
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Full-length cDNA of KIF5B was cloned into a HA-, GFP-, and FLAG-tagged expression vector, pXJ40 (E. Manser, Institute of Molecular and Cell Biology, Singapore). Constructions used in the cellular localization study were DsRed-ER (Clontech), pEYFP-Golgi (#6909-1; Clontech), mRFP-Rab5 (Vonderheit and Helenius, 2005 ; #14437; Addgene), and DsRed-Rab7 (Choudhury et al., 2002 (link); #12661; Addgene). Deletion mutants were generated by PCR using specific primers facilitated by restriction sites. For each construct, several clones were chosen and sequenced to entirety in both directions to confirm their identity. All plasmids were purified using Axygen miniprep kit for use in transfection experiments. E. coli strain DH5α was used as host for propagation of the clones.
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