Supplementary Material
Investigation of membrane protein-protein interactions
using correlative FRET-PLA technique
Daniel Ivanusic1,2, Magdalena Eschricht 1, Joachim Denner1
1
Robert Koch Institute, Berlin, Germany, 2Freie Universität Berlin, Berlin, Germany
BioTechniques 57:188-198 (October 2014) doi 10.2144/000114215
Keywords: : FRET; PLA; protein-protein interaction; HIV-1; CD63
Supplementary Figure 1. Yeast two-hybrid
(Y2H) analysis. CD63 was found as a putative
interaction partner of the viral protein gp41 of
HIV-1 in a split ubiquitin Y2H screen (Y2H
Membrane kit, Dualsystems) against Jurkat
cDNA library with NubG-x orientation. The
columns
present
yeast
(Saccharomyces
cerevisiae strain NMY51) growth on selective
plates leaking the amino acids W, L, H or
nucleotide adenine (A), column 1: positive
control, column 2: co-transformation with bait
and prey containing gp41 and CD63 cDNA
sequence, column 3 and 4: auto-activity of bait
and prey empty vectors, column 5: negative
control. Yeast growth was monitored by
supplementation of single dropout (SD) yeast
media with 3-aminotriazole (3-AT) to increase
interaction stringency. All yeast transformations
were pooled to show interaction on selective
plates.
Supplementary Figure 2. Schematic
presentation of plasmid constructs
expressing fluorescent hybrid proteins.
Boxes represent DNA sequences in the
vectors. CMV: cytomegalovirus promoter,
SP1: cDNA sequence of murine Ig kappachain V-J2-C containing Kozak sequence,
gp41 parts - NHR: N-terminal heptad repeat,
CHR: C-terminal heptad repeat, CFP: cyan
fluorescent
protein,
V5
tag:
GKPIPNPLLGLDST, CD63: cluster of
differentiation 63, YFP: yellow fluorescent
protein, FLAG tag: DYKDDDDK, DNA
sequences are flanked by restriction enzyme
sites used for gene cloning.
Supplementary Figure 3. Workflow for preparation of correlative FRET-PLA samples.
Day 1
seed cells in 6
well plate format
Day 3
wash and fix
samples
cotransfect cells drop cells on polywith YFP and CFP
L-lysine coated
constructs
glass slide
Day 4
proceed extended
washing step
Day 5
CFP/YFP/PLAchannel image
acquisition
block cells
quantify PLA dots
per cell
proceed with
PLA application
correlative
results
48 h
permeabilize,
wash and
block cells
incubate cells with
primary antibody
against tags
store mounted
slides dark at 4°C
incubate
overnight at 4°C
Supplementary Figure 4. Imaging of performed single recognition PLA against FLAG and V5
epitope. HEK293T cells were transiently transfected with pCMV-CD63-YFP, pcDNA-gp41-V5 and empty
vectors pcDNA4B-V5-His, pCMV-Tag2B as controls. Images representing protein expression controls for
single recognition PLA against FLAG and V5 epitopes. Scale bars, 10 µm.
Supplementary Figure 5. Imaging of nuclear localized zinc finger nuclease expression performed by
single recognition PLA against FLAG epitope. HEK293T cells were transiently transfected with pZFNprimer
5´ 3´ sequence
YFP-FLAG (first row) and empty vector pCMV-Tag2B (second row) as control. Scale bars, 10 µm.
Supplementary Table 1. Primer sequences used to generate protein expression constructs.
DI001
TTTTTTGGATCCATGGCGGTGGAAGGAGGAATG
DI002
TTTTTTAAGCTTCATCACCTCGTAGCCACTTC
DI003
TTTTTCTGCAGTTACGCTGACGGTACAGGCCAGA
DI004
TTTTTGCGGCCGCCCTGCCTAACTCTATTCACT
DI005
TTTTTGCTAGCCGCCACCATGGAGACAGACA
DI006
TTTTTGGATCCAGCATAATCTGGAAC
DI007
TTTTTCTCGAGGATGGTGAGCAAGGGCGAGGAGCT
DI008
TTTTTCCGCGGAACCTTTCCGGACTTGTACAGCTC
DI009
TTTTTCTCGAGAAAATGGTGAGCAAGGGCGAGGAGCTG
DI010
TTTTTGGGCCCTTTCTGAGTCCGGACTTGTACAGCTCG
DI011
TTTTCTCGAGAGCTGCCACCATGGTGAGCAAGGGCGAG
DI012
TTTTGGGCCCTTTCCGGACTTGTACAGCTCGTCCAT
DI013
TTTTTCTCGAGAGCGGCCACCATGGTGAGCAAGGGCGA
DI014
TTTTTGGGCCCTTTCTGAGTCCGGACTTGTACAGCTCG