P smad2 3 polyclonal antibody

The third passage of CFs was seeded in a 24-well plate and incubated at 37°C and 5% CO₂. After the cells had been grown to 60% confluence, the medium was removed, and the cells were washed twice with PBS. Then, the cells were treated with 5 ng/mL TGFβ1, followed by culture for 15, 60, or 120 min. In addition, a set of CFs was treated with 10⁻⁶, 10⁻⁵, or 10⁻⁴ mol/L TSN, followed by stimulation with 5 ng/mL of TGFβ1 and further cultured for 120 min. One group with no treatment was used as a control. The experiments in the control group and each treatment group were repeated 3 times. After treatments, cells were stained with 1:100 p-Smad2/3 polyclonal antibody (Abcam, Cambridge, UK) using the SABC immunocytochemical method according to the manufacturer's instructions.[12 (link)]

Total protein was extracted using radioimmunoprecipitation buffer, separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Nanjing Kangruipu Biological Technology Co., Ltd., Nanjing, China) for immunoblotting. The membranes were blocked with 5% defatted milk powder (diluted in TBST) for 1 h and then incubated with rabbit anti-rat FN polyclonal antibody (1:500, Wuhan Boster Biological Engineering Co., Ltd., Wuhan, China) or goat anti-rat Smad2, Smad3, or phosphorylated Smad2/3 (p-Smad2/3) polyclonal antibody (1:500, Abcam, Cambridge, UK) at 4°C overnight. After three washes with PBST, membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (1:5000, Protein Tech Group Inc., Chicago, USA) or rabbit anti-goat IgG (1:9000, Protein Tech Group Inc., Chicago, USA) for 1 h at room temperature. Specific protein bands on the membranes were visualized using enhanced chemiluminescence (Amersham, Piscataway, NJ, USA) according to the manufacturer's instructions, and their absorbance was analyzed using LabWorks 4.5 software (manufacturer: UVP, Upland, USA) and normalized to the β-actin absorbance value.