Non fat dry milk

Anti-α4β7 levels were measured by ELISA. Recombinant
human integrin α4β7 protein (R&D systems) were coated
overnight at 1 ug/mL in PBS in high binding 96 well plates (Costar) and then
blocked with a solution of 1% Tween-20 (Sigma) and 5% non-fat dry milk
(Rockland) in PBS for 2 hours. Serially diluted recombinant
anti-α4β7 Ab (NHP reagents) or plasma samples diluted at
1/5000 were added to the plate for 1h. Plates were then washed five times
with PBS/1% Tween-20 and incubated 1h with mouse anti-monkey IgG-HRP
(Southern Biotech). After five washes, TMB substrate (Southern Biotech) was
applied for 20 minutes. The reaction was stopped with 100 uL/well TMB stop
solution before reading OD at 450nm on an ELISA plate reader (Synergy H4,
BioTek).

Mouse colonic tissues were directly lysed with Triton buffer (0.5%
Triton X-100 and 20 mM HEPES, pH 7.6) on ice, and the lysates were
separated by NuPAGE™ 4–12% Bis-Tris Gel (Invitrogen). Separated
proteins were transferred onto iBlot^® 2 NC Mini Stacks
(Invitrogen) with an iBlot^® 2 Dry Blotting System
(Invitrogen). The filters were then blocked with Tris-buffered saline with 5%
nonfat dry milk (Rockland) plus 0.1% Tween 20 for 1 hour at room
temperature. After blocking, the filters were incubated overnight at
4 °C with primary antibodies (1:1000 dilution) to VDR (sc-13133),
NR1H4 (sc-25309), GPBAR1 (PA5–23182), or actin (sc-8432) and subsequently
washed three times with Tris-buffered saline containing 0.1% Tween 20. The
filters were then incubated with IRDye^® 680LT secondary
antibodies (1:5000 dilution) (LI-COR Biosciences) for 1 hour at room
temperature in the dark. After three washes with Tris-buffered saline containing
0.1% Tween 20, the indicated signals were detected with an
Odyssey^® CLx Fluorescence Imaging System (LI-COR).