Tandem affinity purification of chromatin-remodeling complex protein Baf57c using SGTAP.

<p>(A) Schematic of LR recombination reaction used to create pEpic CMV:Baf57c-SGTAP. (B) Schematic of steps for TAP of Baf57-SGTAP. Step 1: Baf57c with a C-terminally conjugated SBP, TEV protease cleavage site, and tandem copies of protein G is first isolated by affinity purification using IgG-sepharose beads; Step 2: Baf57c-SBP is cleaved from protein G bound to IgG-sepharose beads by the addition of TEV protease; Step 3: Baf57c-SBP is further isolated by affinity purification using streptavidin-sepharose beads; Step 4: Baf57c-SBP is finally eluted from streptavidin by the addition of biotin. (C) Western blot for SBP at various stages of Baf57c purification from nuclear extracts of HEK293T cells expressing pEpic CMV-Baf57c-SGTAP. 10% of each indicated fraction was used for immunoblotting. Lane 1: the crude nuclear extract; Lane 2: nuclear extract after incubation with IgG beads; Lane 3: post-TEV protease cleavage of proteins bound to IgG beads; Lane 4: SDS elution of proteins from beads following Streptavidin purification. The asterisk indicates Baf57c-SGTAP fusion proteins; the arrow indicates the cleaved Baf57c-SBP fusion; molecular weights in kilodaltons are shown at the right.</p

Overview of three-fragment MultiSite Gateway cloning and novel lentiviral destination vectors.

<p>(A) Schematic of an LR recombination reaction and the resulting vector. Site-specific recombination events (red lines) between attR and attL sites from a 5’, middle, and 3’ entry vector with a destination vector replaces the ccdB/Cm^R selection cassette of the destination vector with the mobile DNA elements from the entry vectors, leaving destination vector-specific 5’ and 3’ sequences intact. (B) Schematic of lentiviral destination vectors pEpic and pEpic_Lite. attR3 and 4 sites flanking the ccdB/Cm^R selection cassette are positioned in an anti-sense orientation to viral RNA expression driven by a Rous sarcoma virus (RSV) promoter. pEpic_Lite lacks puromycin resistance (Puro^R). LTR = long terminal repeat; RRE = Rev response element; cPPT = central polypurine tract; ccdB = E. coli ccdB toxin; Cm^R = chloramphenicol resistance; mPGK = mouse phosphoglycerate kinase promoter; WPRE = woodchuck hepatitis virus posttranslational regulatory element.</p

Efficient bicistronic expression from C-terminal P2A conjugation.

<p>(A) Schematic of LR recombination reaction used to create pEpic_Lite mCMV:ErbB3-P2A-GFP. (B) Western blots of COS7 cell lysates 48 hours after co-transfection with ErbB3-P2A-GFP and ErbB2-myc, with or without treatment with neuregulin. Immunoblotting was performed with antibodies against phosphorylated ErbB3 (pErbB3) or GFP.</p

Entry vectors provided in the toolkit.

<p>Entry vectors provided in the toolkit.</p

Overview of pME-BrainbowTEC and application in developing zebrafish spinal cord.

<p>(A) Schematic of pME-BrainbowTEC. (B) No recombination, <i>loxP</i> recombination or <i>lox2272</i> recombination lead to distinct fluorophore expression. (C) Schematic of LR recombination reaction used to create UAS:BrainbowTEC. (D) Possible fluorophore combinations and resulting observed color from three copy expression of UAS:BrainbowTEC. (E) Experimental design for UAS:BrainbowTEC labeling of motoneurons in developing zebrafish spinal cords. Fish carrying multiple copies of UAS:BrainbowTEC were crossed with a dual inducible-Cre and motoneuron-specific GAL4 driver line (<i>mnx1</i>:GAL4; <i>hsp70l</i>:Cre). Embryos were heat-shocked at 7 hours post fertilization (hpf) to induce Cre expression for <i>lox</i> recombination of genomic UAS:BrainbowTEC copies, then imaged at 48 hpf. (F) Representative fluorescent confocal microscope image for GFP, tdTomato, and E2Crimson in 48 hpf zebrafish embryo showing neuronal cell bodies in the spinal cord and motor axons within several adjacent somites. Inset shows neuronal cell bodies in the spinal cord.</p

Effective dual protein expression through N-terminal P2A conjugation to HA-Neuroligin1.

<p>(A) Schematic of LR recombination reaction used to create pEpic_Lite mCMV:memGFP-P2A-HA-Neuroligin1. (B) Dual fluorescent western blot of COS7 cell lysate 24 hours after transfection with mCMV:memGFP-P2A-HA-Neuroligin1. Immunoblotting was performed with antibodies against GFP and HA. (C) Immunocytochemistry for GFP and HA in COS7 cells 24 hours after transfection with mCMV:memGFP-P2A-HA-Neuroligin1. Cells were fixed with paraformaldehyde and surface stained for HA, then permeabilized and stained for GFP. (D) Immunocytochemistry for GFP, HA and the synaptic vesicle-associated protein Synapsin1 in cultured rat hippocampal neurons. Cells were transduced with lentivirus carrying mCMV:memGFP-P2A-HA-Neuroligin1at 2DIV and fixed for immunolabeling at 14DIV. Cells were surface stained for HA, then permeabilized and stained for GFP and Synapsin1. Inset is of an individual basal dendrite segment; the GFP mask is a binarized image of the dendrite using intensity thresholding of the GFP signal. Arrowheads mark dendritic spines containing HA and co-localized Synapsin1 puncta. Scale bar = 10 μm.</p

Rapamycin-induced dimerization to drive nuclear export.

<p>(A) Schematic of LR recombination reaction used to create pEpic CMV:OGT1-mCherry-FRB-HA. (B) Schematic of rapamycin-induced dimerization with FKBP-NES to drive nuclear export. (C) Time-lapse imaging (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159277#pone.0159277.s001" target="_blank">S1 Movie</a>) of mCherry in HEK293T cells transfected with pEpic CMV:OGT1-mCherry-FRB-HA and FKBP-NES. Rapamycin was added at time 0. White arrows mark cell nuclei.</p