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.
Egfp rab4a
Manufactured by Addgene
Sourced in United States, Sweden
EGFP-Rab4A is a plasmid that encodes the early endosome marker Rab4A fused to enhanced green fluorescent protein (EGFP). This construct can be used to visualize the localization of Rab4A, a small GTPase involved in the regulation of early endosome dynamics and recycling.
Automatically generated - may contain errors
Lab products found in correlation
Egfp rab4aq67l, by Addgene (2 mentions)
Egfp rab4as22n, by Addgene (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Mcherry rab5ca, by Addgene (1 mentions)
Mcherry rab5dn, 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)
Mrfp rab5, by Addgene (1 mentions)
Gfp rab7 wt, by Addgene (1 mentions)
Egfp rab7a q67l, by Addgene (1 mentions)
Pha 665752, by Merck Group (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Wortmannin, by Bio-Techne (1 mentions)
Gfp eea1, by Addgene (1 mentions)
Lamp1 rfp, by Addgene (1 mentions)
Pik93, by Bio-Techne (1 mentions)
Gfp p4m sidm, by Addgene (1 mentions)
Gfp rab27a, by Addgene (1 mentions)
4 protocols using egfp rab4a
1
Transient Transfection of Hippocampal Neurons
2
Plasmid Acquisition and Transfection
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All of the plasmids used in the study were obtained from Addgene (Watertown, MA, USA). GFP‐LDLR (#98184; RRID: Addgene_98184) was a gift from Gary Banker & Marvin Bentley (Jenkins et al, 2012 (link)). EGFP‐Rab4a (#49434; RRID: Addgene_49434), EGFP‐Rab4a‐Q67L (#49475; RRID: Addgene_49475), and EGFP‐Rab4a‐S22N (#49476; RRID: Addgene_49476) were gifts from Marci Scidmore (Rzomp et al, 2003 (link)). Rab4a‐T137A mutant was generated using Q5 Site‐Directed Mutagenesis Kit (from New England Biolabs), and the mutation was confirmed by sequencing. Plasmid transfections were performed using Lipofectamine 3000 according to the manufacturer's instructions.
3
Plasmid Database for Cellular Trafficking Studies
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The cDNA encoding Ii (CD74) has been previously described (Bakke and Dobberstein, 1990 (link)). Subcloning and use of the Ii fragment into the pMep4 plasmid has been previously described (Gregers et al., 2003 (link); Skjeldal et al., 2012 (link)). The plasmid encoding mCherry-Rab5 (mCh-Rab5) has been previously described (Haugen et al., 2017 (link)). EGFP-Rab5Q79L was generously provided by Mitsonuri Fukuda (Tohoku University, Miyagi, Japan). pEGFP-Rab7a, pEGFP-Rab7aQ69L and pEGFP-Rab7aT22N were acquired from Cecilia Bucci's laboratory (University of Salento, Lecce, Italy) and have been described previously (Bucci et al., 2000 (link)). The plasmid encoding dsRed-Rab7aT22N was a gift from Richard Pagano (Mayo Clinic and Foundation, Rochester, USA), through Addgene (plasmid #12662 ). The plasmid encoding mApple-Rab7a (Addgene plasmid #54945 ) was a gift from Michael Davidson (Florida State University). EGFP-Rab22 was purchased from Addgene (plasmid #49600 ). The plasmids encoding mRFP-Hrs and EGFP-Eps have been previously described by Haugen et al. (2017) (link). EGFP-Rab4A (plasmid #49434 ) was purchased from Addgene.
4
Visualizing Phosphoinositide Dynamics
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All salts were from Sigma-Aldrich. PAO was from Sigma-Aldrich, and wortmannin and PIK93 were from TOCRIS Bioscience. The following plasmids were used: GFP-PH-OSBP (Balla et al., 2005 (link); gift from Tamas Balla, National Institutes of Health, Bethesda, MD), GFP-PHx2-OSH2 (Stefan et al., 2011 (link); plasmid 36095; Addgene), GFP-P4M-SidM (Hammond et al., 2014 (link); plasmid 51472; Addgene), mRFP-PH-PLCδ1, mRFP-PH-Akt, GFP-Sac2 (Nakatsu et al., 2015 (link); gift from Pietro DeCamilli, Yale University, New Haven, CT), GFP-Rab27a, GFP-Rab3a, NPY-GFP, NPY-mCherry, VAMP2-pHluorin (all gifts from Sebastian Barg, Uppsala University, Uppsala, Sweden), Lamp1-RFP (Sherer et al., 2003 (link); plasmid 1817; Addgene), GFP-Rab5, GFP-EEA1 (gifts from Pietro DeCamilli), CIBN-CAAX (Idevall-Hagren et al., 2012 (link)), GFP-CRY2-5ptaseOCRL (Idevall-Hagren et al., 2012 (link)), GFP-CRY2-iSH2 (Idevall-Hagren et al., 2012 (link)), R-GECO1 (Zhao et al., 2011 (link); plasmid 32444; Addgene), GFP-Lact-C2 (Yeung et al., 2008 (link); plasmid 22853; Addgene), EGFP-Rab4a (Rzomp et al., 2003 (link); plasmid 49434; Addgene), GFP-Rab10 (Huckaba et al., 2011 (link); plasmid 31737; Addgene), Granuphilin-GFP (Gálvez-Santisteban et al., 2012 (link); plasmid 40032; Addgene), and GFP-2xFYVEEEA1 (Petiot et al., 2003 (link)).
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