Colons were excised, opened longitudinally and washed with cold PBS. The
distal-most-3 cm section of each colon was collected and mechanically
homogenized in NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo
Scientific) containing Complete Protease Inhibitor and PhosphoStop (Roche) per
manufacturer’s protocol. Cytosolic and nuclear lysates were subjected to
SDS-PAGE and immunoblotting. The following primary antibodies were use for
immunoblot analysis: anti-pIκBα (Ser32) (14D4) (cat. no 2859);
anti-pIKKα/β (Ser176/180) (16A6) (cat. no. 2697); anti-pp65
(Ser536) (93H1) (cat. no. 3033); anti-NIK (cat. no. 4994); anti-pERK1/2
(Thr202/Tyr204) (D13.14.4E) (cat. no. 4370); anti-p65 (D14E12) (cat. no. 8242);
anti-pSTAT3 (Tyr705) (cat. no. 9131); and anti-STAT3 (79D7) (cat. no. 4904)
(Cell Signaling Technology); anti-p52 (C-5) (cat. no. sc-7386); anti-ERK1 (C-16)
(cat. no. sc-93); anti-ERK2 (C-14) (cat. no. sc-154); anti-CRAMP (G-1) (cat. no.
sc-166055) and anti-Actin-HRP (C-11) (cat. no. sc-1615) (Santa Cruz
Biotechnology); anti-REG3γ (cat. no. ab198216) (Abcam); and anti-Histone
H3 (cat. no. 07-690) (Millipore). Goat anti-rabbit-HRP (cat. no. 111-035-144)
and goat anti-mouse-HRP (cat. no. 115-035-146) (Jackson Laboratories) were used
as secondary antibodies. Protein densitometry were quantified by Image J
software.
distal-most-3 cm section of each colon was collected and mechanically
homogenized in NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo
Scientific) containing Complete Protease Inhibitor and PhosphoStop (Roche) per
manufacturer’s protocol. Cytosolic and nuclear lysates were subjected to
SDS-PAGE and immunoblotting. The following primary antibodies were use for
immunoblot analysis: anti-pIκBα (Ser32) (14D4) (cat. no 2859);
anti-pIKKα/β (Ser176/180) (16A6) (cat. no. 2697); anti-pp65
(Ser536) (93H1) (cat. no. 3033); anti-NIK (cat. no. 4994); anti-pERK1/2
(Thr202/Tyr204) (D13.14.4E) (cat. no. 4370); anti-p65 (D14E12) (cat. no. 8242);
anti-pSTAT3 (Tyr705) (cat. no. 9131); and anti-STAT3 (79D7) (cat. no. 4904)
(Cell Signaling Technology); anti-p52 (C-5) (cat. no. sc-7386); anti-ERK1 (C-16)
(cat. no. sc-93); anti-ERK2 (C-14) (cat. no. sc-154); anti-CRAMP (G-1) (cat. no.
sc-166055) and anti-Actin-HRP (C-11) (cat. no. sc-1615) (Santa Cruz
Biotechnology); anti-REG3γ (cat. no. ab198216) (Abcam); and anti-Histone
H3 (cat. no. 07-690) (Millipore). Goat anti-rabbit-HRP (cat. no. 111-035-144)
and goat anti-mouse-HRP (cat. no. 115-035-146) (Jackson Laboratories) were used
as secondary antibodies. Protein densitometry were quantified by Image J
software.