Anti smad2 antibody

4T1 cells or tumor samples were lysed in TNEN buffer containing proteinase inhibitor cocktail. Proteins were then denatured at 95 °C and loaded onto 12% SDS-PAGE gel, and transferred to PVDF membrane. After incubation with anti-β-Actin antibody (nb100-74340, Novus), anti-TGF-β1 antibody (Abcam, USA), anti-TGFβR1 antibody, anti-Smad2 antibody, anti-Smad3 antibody, or anti-Smad4 antibody (Cell Signaling, USA) at 4 °C overnight, the blots were incubated with HRP-conjugated secondary antibody (1:5000). Signals were visualized using ECL substrates (PerkinElmer, USA) and recorded by UVP BioSpectrum imaging system. The semi-quantitative analyses of the protein levels were performed by Launch VisionWorks LS. β-Actin was used as an internal control for normalization purpose.

Anti-Eed antibody (#09-774, rabbit polyclonal, 1:1,000) was purchased from Millipore. Anti-H3K27me3 antibody (GTX1121184, rabbit polyclonal, 1:500) was purchased from GeneTex. Anti-Actin antibody (I-19, rabbit polyclonal, 1:500) was purchased from Santa Cruz Biotechnology. Anti-Hif1a antibody (NB100-449, rabbit polyclonal, 1:500) was purchased from Novus Biologicals. Anti-Bnip3 antibody (ab10433, mouse monoclonal (ANa40), 2 μg ml⁻¹) was purchased from Abcam. Anti-p-ERK1/2 antibody (#4370, rabbit monoclonal, 1:1,000), anti-p-MEK1 antibody (#9154, rabbit monoclonal, 1:1,000), anti-p-cRaf antibody (#9427, rabbit monoclonal, 1:1,000), anti-p-RSK antibody (#9335, rabbit monoclonal, 1:1,000) and anti-p-p38 antibody (#4511, rabbit monoclonal, 1:1,000), anti-total ERK antibody (#9102, rabbit polyclonal, 1:1,000), anti-p-Smad1/5/8antibody (#9511, rabbit polyclonal; 1:1,000), anti-Smad2 antibody (#5339, rabbit monoclonal, 1:1,000), anti-p-Smad2 antibody (#3104, rabbit polyclonal, 1:1,000), anti-TGF-β receptor II antibody (#3713, rabbit polyclonal, 1:1,000), anti-p-STAT1 antibody (#9171, rabbit polyclonal, 1:1,000) and anti-pSTAT3 antibody (#9145, rabbit monoclonal, 1:2,000) were purchased from Cell Signaling Technology. Western blot analysis was performed according to the standard procedure.

Wildtype and Drak2^-/- CD4⁺ cells were negatively selected with Miltenyi beads and stimulated with 1μg/ml anti-CD3 coated on poly L-lysine-coated coverglass slides and 1μg/ml soluble anti-CD28 for 24 hours. TGF-β was added to some samples for the final 20 minutes. Cells were fixed with 4% methanol-free formaldehyde, permeabilized in 0.1% Triton-X in PBS, washed with PBS, blocked with 1% BSA, and incubated with anti-Smad2 antibody (Cell Signaling) overnight. Cells were stained with Alexa Fluor 647 goat anti-Rabbit, Alexa Fluor 488 Phalloidin, and DAPI (Invitrogen Life Technologies). Images were collected utilizing a Nikon C1Si laser scanning confocal microscope.

Cells were homogenized in RIPA buffer containing 1% protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). The protein levels were quantified using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Amounts of 40 μg of total cell lysates were loaded and separated with 10% SDS-PAGE, and transferred to polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk in TBST buffer at room temperature for 1 h, followed by incubation at 4 °C overnight with the primary antibodies, including an anti-p-Smad2 antibody, anti-p-Smad3 antibody, anti-Smad2 antibody, anti-Smad3 antibody (Cell Signaling, Danvers, MA, USA), and anti-β-actin antibody (Thermo Fisher Scientific, Waltham, MA, USA). Then, the membranes were incubated with horseradish-peroxidase-conjugated IgG secondary antibodies (Thermo Fisher Scientific, Waltham, MA, USA) for 1 h at room temperature. The signals were detected using a chemiluminescence detection kit (Millipore, Burlington, MA, USA). The intensity of the protein was quantified using the ImageJ software.

Cells were lysed in non-denaturing lysis buffer (20 mM Tris HCl pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA and 1% Triton X-100) supplemented with protease (#P8340; Sigma-Aldrich) and phosphatase (#P5726; Sigma-Aldrich) inhibitors. Pre-clearing step of 1 mg cell lysates were performed with 5 µg rabbit IgG (#02-6102; ThermoFisher Scientific) and 20 µl Pierce Protein A/G Magnetic beads (#88802; ThermoFisher Scientific) on a rotating shaker for 1 h and 30 min, at 4 °C. The lysates were then incubated overnight at 4 °C with 5 µg of rabbit IgG or anti-acetylated lysine (#9441; Cell Signaling Technology). The immunoprecipitated proteins were collected after 1h30 incubation with 20 µl Pierce Protein A/G Magnetic beads (4 °C), followed by the elution with 50 µl of Elution buffer (0.1 M Glycine, pH 2.0) and neutralization with a buffer containing 1 M Tris HCl, pH 8.0. Samples were added 1× Laemmli loading buffer β-mercaptoethanol-free, without heating, and separated by SDS-PAGE electrophoresis. As described above, western blotting was performed using anti-Smad2 antibody (#5339; Cell Signaling Technology; 1:1000).