Figure IS-1 Identification of LTA as the bioactive molecule in the peptidoglycan
preparation that is discriminated by oblivious. a-b, Bioactivity and specificity of
peptidoglycan fraction after treatment with a. pronase, proteinase K and lysozyme,
and b. chloroform/methanol extraction (organic phase recovered). c. Dose–response
curve for S. aureus-derived purified LTA. Incubation of inducers with macrophages
was carried out over four hours in each experiment. Data represents mean values /
s.e.m. n=3 mice.
Figure IS-2 Molecular specificity of oblivious-mediated activation by
lipopeptides, and dependency on TLR2. a-d Activation of macrophages derived
from +/+, obl/+ and obl/obl mice by a, R-MALP, b, S-MALP, c, diacylated- and d,
triacylated-Cys-Ser-K4 during a four hour incubation period. e, Production of TNF in
wildtype and TLR2-deficient mice in response to the same ligands during a four hour
incubation period. e, comparison of LTA responses between peritoneal macrophages
derived from wildtype (F2 C57BL/6/129SV/J), oblivious (obl/obl), and Tlr2/ mice
and Scarb1/ mice. Data represents mean values / s.e.m. n=3 mice.
Figure IS-3 The oblivious mutation only partially blocks activation of MAP
kinases and IB degradation when cells are stimulated with LTA (a) or racemic
MALP-2 (b). western blot analysis of JNK, p38, and ERK1/2 phosphorylation, and
degradation of IB in macrophages from normal C57BL/6 mice (+/+) as well as obl
homozygous mutants (obl/obl) when cells were stimulated with LTA (100 ng/ml).
Data are representative of three independent experiments.
Figure IS-4 Spontaneous eye infection in oblivious mice. C57BL/6 wildtype (a) and
obl/obl (b) mice at the age of 6 months. A semilunar pterygium gradually extends to
cover the surface of the eye. (c) Development of endophthalmitis is frequently
observed in oblivious mice by one year of age. (d) Colonization of the surface of the
eye by Gram positive bacteria (Corynebacterium species and Staphylococcus lentus)
in control mice and obl/obl mice (n=10 mice/group with age >6 months; error bars
indicate s.e.m).
Figure IS-5 Western blot analysis of CD36 in cell extract or supernatant from
wildtype or oblivious mice cultured for 24 h in vitro.
Figure IS-6 CD36 augments the response to LTA and R-MALP-2. HEK293 cells
were transfected with either control vector, CD36, TLR2, and/or TLR6 in
combination with a NF-B luciferase reporter gene as indicated. After overnight
incubation, cells were stimulated with different concentrations of LTA (a), S-MALP
(b) or PAM3CSK4 (c) after which luciferase activity was measured. Data are derived
from triplicate transfections, and error bars indicate s.e.m. Significant augmentation of
LTA and S-MALP (but not PAM3CSK4) sensing is conferred by CD36, both in the
presence and in the absence of TLR6, which also augments sensing. P-values reflect
pairwise comparisons by ANOVA.
Figure IS-7 Unrooted tree depicting phylogeny of the CD36 family. CD36
homologues were identified by blastp searches of the GenBank nr database and the
Celera database (human, mouse and Drosophila). The tree was produced using
ClustalW. Droso, Drosophila melanogaster. Croquemort is indicated by a blue square
for reference. Note that CD36 has two paralogs in humans and in mice (SR-B1 and
SR-B2), each represented in other vertebrates as well. However, no orthologous
relationship is observed between CD36, SR-B1, or SR-B2 and any invertebrate
sequence.