Anti smad7 antibody

According to the needs of different Co-IP experiments, related proteins were overexpressed through gateway system. Briefly, cell lysates were collected using ice-cold IP lysis buffer. The lysates were transferred to a micro centrifuge tube and centrifuged at 13,000×g for 10 min. The supernatant was transferred to a new tube for protein concentration determination and further analysis. The Co-IP analyses were performed using a Co-Immunoprecipitation Kit (ThermoFisher Scientific, #26149) according to the manufacturer's protocol. The experimental steps including: pre-clear lysate using the control agarose resin, Co-IP, elution of Co-IP, resin regeneration and preparation for SDS-PAGE analysis were carried out in turn. Antibodies used therein include: anti-HDAC5 antibody (Invitrogen, PA1-41117, 1:500), anti-Smad7 antibody (Santa Cruz, sc-365846, 1:200), anti-MEF2A antibody (Santa Cruz, sc-17785, 1:200) and anti-NF-κB p65 antibody (Santa Cruz, sc-8008, 1:200). Normal rabbit IgG without antigenicity provided with the kit was used as a negative control. SDS-PAGE and immunoblotting were performed as described above.

Cervical cancer samples and cells were homogenized using a lysis buffer containing 50 mM Tirs-Cl, pH 7.4, 120 mM NaCl, 1 % NP-40, 0.2 % SDS, 1 mM EDTA and complete protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland), and centrifuged for 20 min at 13,000g, 4 °C. The protein concentration of cell lysate was analyzed using BCA protein assay kit (Bio-Rad, Hercules, CA). The protein samples were separated by SDS-PAGE and transferred into PVDF membranes. The membranes were blocked using blocking solution (150 mM NaCl, 20 mM Tris, pH 8.0, 0.05 % Tween-20, 5 % non-fat milk). Thereafter, the membranes were incubated with the indicated primary antibodies: rabbit polyclonal anti-Smad7 antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal anti-GAPDH antibody (TA-08, Zhongshan Golden Bridge, Beijing, China). Secondary antibody incubation was conducted using horseradish peroxidase (HRP)-conjugated goat anti-mouse and anti-rabbit antibodies. The protein bands were visualized using ECL methods according to the manufacturer’s instruments (Zhongshan Golden Bridge, Beijing, China). The densities of protein bands were relatively quantified using ImageJ software (WS Rasband, ImageJ, NIH, Bethesda, MD). All experiments have been independently performed for three times.