Ibright fl1000 imager

Flag-tagged TGF-βs and HA-tagged LRRC33 or GARP-Expi293F
transfectants (~1×10⁷/ml) were lysed at 4°C for
30 minutes in 500 μl of 150 mM NaCl, 50 mM Tris-HCL pH8.0, 1% Triton-x100
(Sigma-Aldrich), 0.1% SDS (Sigma-Aldrich), 0.5% sodium deoxycholate, 1mM EDTA,
and protease inhibitor cocktail (Roche) and centrifuged at 14,000 rpm at
4°C for 5 minutes. For IP, cell lysates (~400 μL) were
incubated with 2 μg/mL of anti-Flag M2 (Sigma, F1804) overnight at
4°C with rotation and then 10 μL of protein G Sepharose beads (50%
suspension in PBS) (GE Healthcare) was added for 2 hours at 4°C with
rotation. For WBs, 12.5 μl of 5X SDS sample buffer was added to 50
μl of cell lysate or 60 μl of 1X SDS sample buffer was added to
pelleted, washed beads from IP. Samples were heated at 95°C in sample
buffer containing 5 mM N-ethylmaleimide (Calbiochem) or 5%
β-mercaptoethanol and 12 μl per lane was subjected to non-reducing
and reducing SDS-polyacrylamide gel electrophoresis (PAGE), respectively.
Proteins were transferred to PVDF membrane (Bio-Rad) using Trans-Blot Turbo
System (Bio-RAD) and probed with 2 μg/mL of specific antibodies
(anti-Flag M2, Sigma F1804; anti-HA, 12CA5), and detected with 1 μg/mL of
HRP-conjugated secondary antibodies (Abcam, ab6789). Blots were developed using
the SuperSignal West Pico Plus Chemiluminescent Substrate (Thermo Fisher) and
were imaged using an iBright FL1000 imager (Invitrogen).

Tissues were homogenized in RIPA buffer (Sigma). Equal amounts of protein were loaded into a 4%-12% NUPAGE gel (Fisher). The proteins in the gel were transferred onto PVDF membranes (Invitrogen) using the Invitrogen iBlot system. The top half of the membranes (containing IgG) were blocked overnight at 4°C in 5% Blocking Grade Blocker (Bio-Rad) and 0.1% Tween-20 (Bio-Rad) in PBS (VWR). The membrane was then incubated with goat anti-Rat IgG-HRP (Bio-Rad, 5204-2504, 1:10,000) for 1 hour at room temperature. The lower half of the membrane (containing GAPDH) was blocked for 2 hours at room temperature and incubated overnight at 4°C with the primary antibody, mouse anti-GAPDH (6C5) (Calbiochem, 1:10 000). After washing, the membrane was incubated 30 minutes with the secondary antibody, goat anti-Mouse-HRP (Cell Signaling, 7076P2, 1:10 000). After the last antibody incubation, both membranes were washed and incubated with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, 34095) and imaged using an iBright FL1000 imager (Invitrogen).
Plasma zonulin, a marker for gut barrier integrity, was measured using a rat zonulin ELISA kit (MyBioSource, MBS753035) according to the manufacturer’s protocol.