Supplementary Information #1
Expression of GFP correlates with expression
of TLR2. RAW TT10 cells were transfected with
wild type HA-tagged TLR2-TIGZ2+ as in other
figures in the manuscript. After 24 hours, the
cells were stained with a mouse monoclonal
antibody to the extracellular HA epitope (Babco)
followed by a Goat-anti-mouse IgG1 antibody
coupled to phycoerythrin (PharMingen). The
cells were analyzed by flow cytometry. The level
of surface expression of TLR2 correlated directly
with the level of GFP expression from the
bicistronic message. The range of GFP detection
was greater than the range of HA-TLR2
detection. Thus, gating on GFP-hi cells (green
box) facilitated inclusion of TLR2-expressing cells and exclusion of non-expressing
cells.
Inh
ibition of zymosan-induced TNF- production is observed if TLR2-P681H
expression is monitored by surface HA epitope staining. RAW TT10 cells were
transiently transfected with HA-tagged TLR2 in the bicistronic expression vector
(TIGZ2+, left panel), or in pDisplay (right panel). 24 hours after transfection the cells
were stimulated with zymosan for 6 hours, and TNF- production was detected with an
antibody to mouse TNF- as described in the manuscript. In the left panel TLR2P681H-expressing cells were detected by GFP expression (using a gate as illustrated
above in green) demonstrating (as described in the manuscript) that cells expressing
the inhibitory receptor (thick green line) produced TNF-, while cells not expressing the
receptor (thin green line) failed to produce the cytokine. When surface TLR2-P681H
expression was monitored by surface HA staining (using a gate as illustrated above in
red), zymosan-induced TNF- production was inhibited in cells expressing the inhibitory
receptor (thick red line) as compared to cells not expressing the receptor (thin red line).
In both panels, background TNF- production is indicated with a black line. The effect
of the inhibitory receptor is consistently more evident on GFP-gated cells compared to
HA-gated cells due to the greater exclusion of non-expressing cells using the GPF
method.