Horseradish peroxidase conjugated goat anti rabbit secondary antibodies

Total protein of the cells was isolated with RIPA tissue lysis Buffer (Solarbio, Beijing, China) in accordance with the operating manual. The concentration of the obtained protein was measured with BCA. Equal amounts (50 mg) of protein were separated on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Thermo Scientific). Membranes were incubated with primary rabbit antibodies against ANLN antibody (1 : 3000, 66643-1; Proteintech), RhoA (1 : 1000, 2117; Cell Signaling Technology, Danvers, MA, USA), and β-actin (1 : 5000, AC026; ABclonal) at 37°C overnight. The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1 : 10000; ABclonal) for 1 h at room temperature. The signals were detected with an enhanced chemiluminescence detection reagent (Thermo Scientific). The bands were quantified via densitometry and analyzed with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Relative protein levels were normalized to β-actin.