Biosci. Rep. (2012) / 32 / 333–343 (Printed in Great Britain) / doi 10.1042/BSR20110100
SUPPLEMENTARY ONLINE DATA
Live-cell visualization of transmembrane protein
oligomerization and membrane fusion using
two-fragment haptoEGFP methodology
Derek J. QUINN*, Neil V. MCFERRAN*, John NELSON* and W. Paul DUPREX†1
*School of Biological Sciences, Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, U.K., and †Department of Microbiology,
Boston University School of Medicine, Boston, MA 02118, U.S.A.
Figure S1 Quantification of the fluorescence signal from Vero
cells transfected with pN157 (6)zip and pzip(4)C158 haptoEGFP
split point constructs fused to known interacting leucine zippers
(A) Generated fluorescence was read in a Tecan fluorescent plate reader
(excitation, 485 nm and emission, 535 nm). The results are represented as equivalent sgGFP expressed in femtomoles; also shown are negative controls demonstrating that the generated fluorescent signal from
reconstituted haptoEGFP fragments is driven only by the specific interaction of the leucine zippers. (B) Corresponding bar chart showing the
quantified fluorescent signal for Vero cells transfected with pN172 (6)zip
and pzip(4)C173 haptoEGFP split point constructs.
1
To whom correspondence should be addressed (email pduprex@bu.edu)
www.bioscirep.org / Volume 32 (3) / Pages 333–343
D. J. Quinn and others
Figure S2 Immunoblotting of Vero cells transfected with various
complementary haptoEGFP constructs spilt at several different
loop regions and fused to a known interacting pair of leucine
zippers
Detection of expressed proteins was carried out by immunoblotting
with a mixture of two anti-EGFP rabbit polyclonal antisera. (A) HaptoEGFP construct pN142 (6)zip- and pzip(4)C143 + -transfected cells. Lane
1, SeeBlue plus 2 molecular-mass markers; lane 2, mock-transfected
Vero cells; lane 3, hapto fusion construct pN142 (6)zip-transfected cells;
lane 4, hapto fusion construct pzip(4)C143 -transfected cells); lane 5,
hapto fusion constructs pN142 (6)zip and pzip(4)C143 -transfected cells;
lane 6, pNPSA(EGFP)-transfected cells; lane 7, recombinant MVeGFP
virus-infected cells; lane 8, SeeBlue plus 2 molecular-mass markers.
(B) Hapto construct pN157 (6)zip- and pzip(4)C158 -transfected cells. Lane
1, SeeBlue plus 2 molecular-mass markers; lane 2, mock-transfected
Vero cells; lane 3, hapto fusion construct pN157 (6)zip-transfected cells;
lane 4, hapto fusion construct pzip(4)C158 -transfected cells; lane 5,
hapto fusion constructs pN157 (6)zip and pzip(4)C158 -transfected cells;
lane 6, pNPSA(EGFP)-transfected cells; lane 7, recombinant MVeGFP
virus-infected cells; lane 8, SeeBlue plus 2 molecular-mass markers.
(C) Hapto construct pN172 (6)zip- and pzip(4)C173 -transfected cells. Lane
1, SeeBlue plus 2 molecular-mass markers; lane 2, mock-transfected
Vero cells; lane 3, hapto fusion construct pN172 (6)zip-transfected cells;
lane 4, hapto fusion construct pzip(4)C173 -transfected cells; lane 5,
hapto fusion constructs pN172 (6)zip- and pzip(4)C173 -transfected cells;
lane 6, pNPSA(EGFP)-transfected cells; lane 7, recombinant MVeGFP
virus-infected cells; lane 8, SeeBlue plus 2 molecular-mass markers.
(D) Hapto construct pN190 (6)zip- and pzip(4)C191 -transfected cells. Lane
1, SeeBlue plus 2 molecular-mass markers; lane 2, mock-transfected
Vero cells; lane 3, hapto fusion construct pN190 (6)zip-transfected cells;
lane 4, hapto fusion construct pzip(4)C191 -transfected cells; lane 5,
hapto fusion constructs pN190 (6)zip- and pzip(4)C191 -transfected cells;
lane 6, pNPSA(EGFP)-transfected cells; lane 7, recombinant MVeGFP
virus-infected cells; lane 8, SeeBlue plus 2 molecular-mass markers.
Figure S3 Quantification of the fluorescence signal from Vero
cells transfected with various MV-H and MV-F haptoEGFP fusion
constructs
Generated fluorescence signal was read on a Tecan fluorescent plate
reader (excitation 485 nm, and emission 535 nm). The results are represented as equivalent sgGFP expressed in femtomoles. Also shown
are negative controls where complementary MV-H or MV-F haptoEGFR
fusion constructs have been replaced with those fused to a leucine
zipper, demonstrating that the fluorescent signal is generated only by
the specific interaction of the MV glycoproteins in either homo-oligomeric or hetero-oligomeric complexes. The final bar shows the positive
control level of fluorescence obtained with two complementary leucine
zipper fusions. The increased level of fluorescence observed with the
combined MV-H/MV-F haptoEGFP constructs, as compared with the experiments involving a single viral protein fusion, reflects the potential
for viral-protein-driven non-fluorogenic homotrimerization of non-complementary haptoEGFPs, such as trimeric [N157 (16)H]3 . In the case of the
N157 (16)H/F(14)C158 experiment, all H–F glycoprotein associations will
lead to fluorescence.
Received 30 August 2011/22 November 2011; accepted 2 March 2012
Published as Immediate Publication 2 March 2012, doi 10.1042/BSR20110100
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