Anti smad7

Cells from untreated group, TGF-β1-treated and SB431542-treated cells were washed 3 times with ice-cold PBS. Lysis buffer was then added, and the cells were lysed on ice for 30 min, followed by centrifugation at 12,000 rpm for 10 min at 4°C to remove cell debris. Protein samples were mixed with loading buffer and heated at 100°C for 10 min, after which the samples were separated by 10% SDS-PAGE gels (Bio-Rad, California, USA). Proteins were then transferred to polyvinyl difluoride membranes (Solarbio Systems, Beijing, China). Membranes were blocked in 5% nonfat milk in TBST buffer for 1–2 h at room temperature. The blots were incubated with primary antibodies overnight at 4°C with rabbit anti-p-Smad2 (1∶1000, Cell Signaling Technology, Massachusetts, USA), anti-Smad7 (1∶200, Santa Cruz Biotechnology, California, USA), anti-E-cadherin (1∶500, Abcam, Cambridge, United Kingdom), mouse anti-vimentin (1∶100, Santa Cruz Biotechnology, California USA), or anti-N-cadherin antibodies (1∶1000, Abcam, Cambridge, United Kingdom)This procedure was followed by incubation with sheep anti-mouse (1∶20000) or anti-rabbit (1∶20000) HRP-labeled secondary antibodies for 2 h at room temperature. The bands were detected with an ECL reagent kit (Thermo Systems, Massachusetts, USA). β-Actin immunoblots served as a loading control. All experiments were repeated three times.

For immunoprecipitation, the protein lysates were prepared from control LPMC treated with Ex527 or DMSO for 8 h and IBD LPMC treated with Cay10591 or DMSO. Proteins were immunoprecipitated with 2 μg/500 μl of anti-Smad7 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or control isotype antiserum for 2 h followed by incubation with protein A/G agarose beads overnight. The resulting immunoprecipitates were washed four times with cold lysis buffer, separated by SDS-polyacrylamide gel electrophoresis, and immunoblotted with antibodies against acetyl-lysine (final dilution 1:2,000, Cell Signaling Technology, Danvers, MA, USA) followed by a horseradish peroxidase-conjugated anti-rabbit IgG antibody (Promega, Madison, WI USA) or with antibody against ubiquitin (final dilution 1:400, Santa Cruz Biotechnology) followed by a horseradish peroxidase-conjugated anti-mouse IgG antibody. The reactions were detected and analyzed as above. At the end, the blots were stripped and incubated with anti-Smad7 antibody (final concentration 1 µg/ml, R&D Systems).

Western blot analyses were performed as described previously [7 (link)]. Briefly, samples (20 μg protein) were subjected to SDS–PAGE. The proteins were transferred onto nitrocellulose membranes, which were probed with the specific antibodies: anti-ZO-1, anti-vimentin, anti-Smad7 and anti-Smurf2 (Santa Cruz, 1:1000), anti-pan-Akt (phospho T308) antibody (Abcam) also known as anti-pAkt, anti-total Akt, (Cell Signaling, 1:1000) and anti-FN (BD Biosciences, 1:1000). A peroxidase-conjugated goat anti mouse IgG (1: 10 000) was used as a secondary antibody. For detection of other proteins or β-actin, the membranes were treated with Stripping buffer and then re-probed with another antibody or β-actin antibody, the latter served as an internal control.

Prepared heart tissue and cell samples were solubilized in immunoprecipitation lysis buffer (20 mM Tris pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 5% glycerol, protease inhibitor cocktail) and precipitated by centrifugation. The prepared tissue and cell supernatants (800 μg) were incubated with anti-CAR3 (Santa Cruz, 1μg per 250 μg of total protein) antibody overnight at 4°C followed by incubation with 30 μL of protein G Agarose beads (Santa Cruz) for 4 hours at 4°C. After being washed three times with cold wash buffer and once with lysis buffer, the immunoprecipitations were resuspended in 30 μL of loading buffer and subjected to western blot for examination of Smad7. Following consecutive washes, the immunoprecipitations containing the CAR3-bound or IgG control-bound proteins were resuspended in 30 μL of loading buffer and subjected to western blot for examination of Smad7. Whole lysate/input sample was used as a control of the immunoprecipitation enrichment. Then, tissue and cell lysates (800 μg) were prepared and carried out by incubation with anti-Smad7 ((Santa Cruz, 1 μg per 250 μg of total protein) antibody overnight at 4°C followed by addition of 30 μL of protein G Agarose beads. The eluted samples were further analyzed via western blotting to detect the Ac-lysine modification of Smad7.