Enhanced chemiluminescence ecl luminous fluid

Chicken uterine gland cells were seeded in 6-well dishes and cultured in growth medium, and were transfected when the confluence reached 70 to 80%. After 48 h of transfection, total protein was extracted using a total protein extraction kit (Solarbio, Beijing, China) and the protein concentration was determined using a BCA protein determination kit (BestBio, Shanghai, China) according to the manufacturer's instructions. Equivalent amounts of total proteins from the different treatments were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with QuickBlockTM blocking buffer (Beyotime, Shanghai, China) for 2 h at room temperature. The membranes were incubated with specific primary antibodies at 4°C overnight, including anti-CA2 and anti-β-tubulin (Abcam, Cambridge, UK; diluted 1: 1,000). The next day, the PVDF membranes with proteins were incubated with a specific secondary antibody (ZenBio, Chengdu, China; diluted 1:5,000). Finally, enhanced chemiluminescence (ECL) luminous fluid (Beyotime) was used for detection. The relative gray scales of CA2 were detected using ImageJ software, and 3 independent replicates were set for each treatment.