Nondenaturing lysis buffer

Non-denaturing proteins of PK cells and LM of the pig were extracted by Non-denaturing Lysis Buffer (Sangon). Later, we typically bonded non-denaturing proteins and biotin-labeled DNA probes by rotation, which were then supplemented with Streptavidin MagneSphere^® Paramagnetic Particles (Promega). Then, the reactions were further rotated and washed. Later, DNA-bound proteins were collected with 10% SDS (sodium dodecyl sulfate, sodium salt) and analyzed by Western blotting, taking non-denaturing proteins/Streptavidin MagneSphere^® Paramagnetic Particles as positive/negative control.

Total protein was isolated with radioimmunoprecipitation assay (Sigma–Aldrich) buffer supplemented with Protease Inhibitor Cocktail (Roche). Lysates were separated from the supernatant by centrifugation. Proteins were separated using SDS–polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane. For native PAGE, proteins were extracted with Nondenaturing Lysis Buffer (Sangon Biotech, C510013) and separated by native PAGE on a precast GLgel with HEPES Native‐PAGE (BBI, C601100). The samples were successively combined with primary antibodies and secondary antibodies conjugated with HRP. The secondary antibodies were PSD95 (rabbit, 1:1000; #3409, CST), anti‐rabbit IgG (H + L) (1:5000; #14708, CST), and goat anti‐rabbit IgG (1:5000; A0208, Beyotime).

Marc-145 cells cultured in 10-cm-diameter dishes were collected and lysed in nondenaturing lysis buffer (Sangon, Shanghai, China) supplemented with protease and phosphatase inhibitor cocktails. An equal mass of lysate was incubated overnight with 2 μg of anti-ARID3A (A7668, Abclonal, Wuhan, China), anti-p65 (A11201, Abclonal, Wuhan, China) or anti-IgG (Beyotime, Jiangsu, China) together with 25 μl of Protein A+G Agarose beads (Beyotime, Jiangsu, China). After centrifugation, the beads were washed with 1 mL of lysate buffer three or four times. Then, 15 μl of 2×SDS loading buffer was added to the beads, boiled for 10 min, and loaded onto an SDS–PAGE gel for western blot analysis.