Hrp conjugated goat anti rabbit igg h l

According to Menicacci, B. et al. [17 (link)], five groups of cells were treated with drugs, and the cells were harvested after 24 h of culturing. Radioimmunoprecipitation assay buffer (RIPA buffer) (Bioworld technology, St. Louis Park, MN, USA)-induced lysis was performed for 30 min, and the supernatant was collected after centrifugation at 12,000 rpm for 5 min. The protein content of the lysates was measured using a BCA protein assay kit (Biotopped, Beijing, China) following the manufacturer’s protocol. Equal amounts of protein standard solutions were loaded into the corresponding lanes, run under 10% SDS polyacrylamide gel electrophoresis, then transferred to polyvinylidene fluoride membranes and blocked for one hour at room temperature (25–36 °C). The membranes were then mixed with specific primary antibodies—anti-TGF-β1 (1:2000), anti-Smad3 (1:2000), anti-phospho-Smad3 (Ser425) (1:2000), anti-Smad4 (1:2000), Type I collagen (1:1000), and anti-β-actin (1:5000) antibodies—overnight at 4 °C. The membrane was then incubated with secondary antibody HRP-conjugated Goat Anti-Rabbit IgG(H+L) (1:5000, Proteintech Group, Wuhan, China) for one hour at room temperature. Protein bands were visualized using an ECL Western Blot Detection Kit (1:5000, Gsebio, Xi’an, Shanxi, China).