protein G plus; PIERCE, respectively). The albumin- and IgG-depleted sera were
analyzed by SDS/PAGE, 2-DE, and WB analysis.
Human HPs. HP1–1 and HP2–2 extracted from human plasma were purchased
from Sigma. HP SDS/PAGE, both monodimensional gel electrophoresis and
2-DE, WB, and MS analyses are described in detail in (SI Text). HP deglycosy-
lation was performed by addition of PNGase F according to the manufactur-
er’s instructions (Sigma).
Human Zonulin/Pre-HP2 Cloning and Expression in a Baculovirus Expression
System and Its Cleavage by Proteases. Recombinant zonulin/preHP2 protein
production using a baculovirus system and its puriﬁcation are described in SI
Text. Puriﬁed single-chain zonulin was subjected to proteolytic cleavage using
the serine proteases indicated, resolved by SDS/PAGE, and then stained with
SimplyBlue SafeStain solution (Invitrogen). For generation of 2-chain HP2,
single-chain zonulin was exposed to trypsin-agarose beads (T-1763; Sigma) for
20 min at 25 °C. The beads were removed by centrifugation, and the effec-
tiveness of the removal of trypsin was conﬁrmed by assay of trypsin peptidase
activity against the substrate Glu-Gly-Arg-pNA (Bachem BioScience).
Ex Vivo and In Vivo IP Studies. The effects of zonulin on ex vivo and in vivo IP
were determined as previously described (8, 14) and are reported in detail in
Zonulin Activation of EGFR. To determine whether zonulin can activate EGFR,
increasing concentrations of either zonulin or 2-chain mature HP2 were added
for increasing exposure times to serum-starved high EGFR-expressing Caco-2
cells. The cells were lysed and processed for WB analysis with anti-phospho
EGFR (Y1068) Ab (Cell Signaling Technology, Inc.) as previously reported (39).
Experiments were repeated in the presence of 5
M of the EGFR-selective PTK
inhibitor AG1478 (Calbiochem).
Knockdown of PAR2 Through RNA Interference. The methods used to silence
PAR2 are reported in detail in SI Text.
Zonulin Gene Sequencing and Quantification from Intestinal Tissue from Pa-
tients With and Without CD. Samples of small-intestine mucosae were obtained
from the second or third portion of the duodenum from subjects undergoing
a diagnostic upper gastrointestinal endoscopy. Subjects included were 10
healthy controls, 7 patients with active CD at diagnosis, and 3 patients with CD
on treatment with a GFD for at least 6 months. All patients had clinical
indications for the procedure and gave their informed consent to undergo an
additional biopsy for the purpose of this study. The study protocol was
approved by the Ethics Committee of the University of Maryland. The small-
intestine biopsies were immediately collected in RNAlater RNA Stabilization
Reagent (Qiagen) and stored at ⫺20 °C until processed. Total RNA extraction,
cDNA synthesis, and real-time PCR are described in SI Text.
Statistical Analysis. All values are expressed as mean ⫾ SE. The analysis of
differences was performed by 2-tailed Student’s t tests to test differences
between 2 groups for either paired or unpaired varieties. Multivariate analysis
was performed where appropriate. Values of P ⱕ 0.05 were regarded as
ACKNOWLEDGMENTS. This manuscript was partially supported by National
Institutes of Health grant DK048373 (to A.F.).
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