Protein Extraction and Western Blotting

Protein preparation and western blotting were conducted as described previously [53 (link)]. Briefly, the MO with TNC was collected and lysed in RIPA buffer containing phenylmethanesulfonyl fluoride and PhosSTOP (Solarbio, China). The supernatant containing proteins was collected and stored at − 80 °C until use. Protein concentrations were measured with a BCA kit (Beyotime, China). Equivalent amounts of the proteins were separated on SDS–PAGE gels and transferred to polyvinylidine difluoride membranes (Bio-Rad, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 (0.5%) for 2 h. Subsequently, the membranes were incubated with the primary antibody at 4 °C overnight and incubated with the secondary antibody at 37 °C for 2 h. The HRP ECL system (Beyotime, China) was used to visualize the protein bands, and the gray values were then analyzed.