Mab2029

The sections of fresh surgical specimens taken from the neoplastic areas and the normal, adjacent colonic mucosa of CRC patients and CRC cell lines were fixed with paraformaldehyde (4% final concentration), permeabilized with Triton X-100, and then blocked at room temperature with BSA 1%, Tween 0.1%, and glycine 2%. After overnight incubation at 4 °C with a rabbit primary antibody against p-Stat3 Tyr705 (1:50, catalog number 9145, Cell Signaling Technology, EuroClone, Milan, Italy) or mouse primary antibody against Smad7 (1:50, catalog number MAB2029, R&D Systems), sections were treated with a secondary antibody goat anti-rabbit Alexa488 (1:1500, catalog number A11008, Invitrogen, Waltham, MA, USA) or goat anti-mouse Alexa488 (1:1000, catalog number A11017, Invitrogen) for 1 h at room temperature. Slides were finally mounted using the ProLong gold antifade reagent with DAPI (catalog number P36931, Invitrogen), and analyzed with a LEICA DMI4000 B microscope, using LEICA application suite software (V4.6.2) (Leica, Wetzlar, Germany).

RIPA buffer (Life Technologies, Inc., 89901) with phosphatase inhibitor and protease inhibitor (Roche Applied Science) was used to lyse cells. Cell lysates were sonicated with a Branson sonifier. Protein concentration was measured with protein assay (Bio-Rad) with Spectrophotometer 3000 (Bio-Rad) in duplicates. 10–50 μg of protein was loaded in NuPAGE 4–12% BisTris gel for protein fractionation, and protein was then transferred to nitrocellulose membrane (VWR, 732-3031). After blocking, protein was probed with primary antibody followed by detection with horseradish peroxidase-conjugated secondary antibody (Dako) after washing. The following primary antibodies were used: calponin (ab46794, Abcam); SM22 (ab14106, Abcam); αSMA (A2547-100, Sigma); SMMHC (ab53219, Abcam); SMAD7 (MAB2029, R&D Systems); and ROCK2 (ab71598, Abcam).

Duodenal samples were lysed on ice in buffer containing 10 mM HEPES [pH 7.9], 10 mM KCl, 0.1 mM EthyleneDiamineTetraacetic Acid (EDTA), 0.2 mM Ethylene Glycol-bis (β-aminoethyl ether)-N,N,N',N'-Tetraacetic Acid (EGTA), and 0.5% Nonidet P40 supplemented with 1 mM dithiothreitol (DTT), 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM Na₃VO₄, and 1 mM NaF. Lysates were clarified by centrifugation and separated on sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. Blots were incubated with antibodies against SMAD7 (1 μg/ml MAB2029, R&D Systems, Minneapolis, MN, USA), total ERK (sc-94, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-SMAD2,3 (#8828, Cell Signaling Technology, Danvers, MA, USA), total and phosphorylated STAT4 proteins (sc-486, sc-28296, Santa Cruz Biotechnology). Densitometry data for SMAD 7 and SMAD2/3 was normalized using ERK1/2 by calculating values expressed as arbitrary units (a.u.) using ERK1/2 as the denominator. Multiple Western blots were performed (blots were stripped and then incubated with different antibodies), and densitometry data from each Western blot were taken individually for analysis given inherent variability in densitometry as is established [24 (link)]. Limited availability of tissue samples made it impractical to perform transcriptional-level analyses using RNA.