The cells were extracted with protein lysis buffer (Beyotime, China) supplemented with protease inhibitor cocktail. Protein concentration was determined using the BCA Kit (Beyotime, China). Proteins (25–35 μg) were separated on a 10% polyacrylamide precast SDS gel (Bio-Rad, Richmond, CA, USA) followed by blotting on PVDF membranes (Millipore Billerica, MA, USA). For IP experiments, cells were lysed in IP buffer (Beyotime, China) and incubated with IP-grade antibodies followed by the pull-down with protein A/G beads (161-4023) (Bio-Rad, Richmond, CA, USA) for subsequent immunoblot analyses.
For pull-down assay, MODE-K cell lysates were collected and centrifuged at 8000 × g. The supernatant was transferred to another tube, and the cell debris was thoroughly discarded. Prewashed streptavidin beads were added into the supernatant, allowing 2 h preincubation with motion at 4°C to remove unspecific binding proteins. Purified mouse recombinant Nur77-LBD proteins were dissolved in lysis buffer. The lysates or recombinant proteins were incubated with compounds, followed by incubation with indicated doses of biotin-GPA for 6 h. Beads were washed with IP buffer for three times and boiled in SDS buffer.
For pull-down assay, MODE-K cell lysates were collected and centrifuged at 8000 × g. The supernatant was transferred to another tube, and the cell debris was thoroughly discarded. Prewashed streptavidin beads were added into the supernatant, allowing 2 h preincubation with motion at 4°C to remove unspecific binding proteins. Purified mouse recombinant Nur77-LBD proteins were dissolved in lysis buffer. The lysates or recombinant proteins were incubated with compounds, followed by incubation with indicated doses of biotin-GPA for 6 h. Beads were washed with IP buffer for three times and boiled in SDS buffer.