Protein a g plus agarose

ACC was rapidly removed and washed in ice-cold PBS (0.1 mM) and lysed in NP-40 lysis buffer (Beyotime). The extract was pre-incubated with protein A/G PLUS-Agarose (Thermo Fisher) and normal rabbit IgG antibodies for 30 min at 4 °C. The mixture was then centrifuged at 2500 rpm for 5 min at 4 °C. For immunoprecipitation, equal amounts of protein were incubated with anti-p65, anti-LXRβ, or rabbit IgG at 4 °C for 2 h. Then, resuspended magnetic beads were added to each sample and incubated overnight at 4 °C. Magnetic beads were washed with lysis buffer three times and resuspended in SDS sample buffer. The immunoprecipitated protein complex were separated by SDS-PAGE and analyzed by immunoblotting using antibodies against p65 or LXRβ.

Immunoprecipitation and immunoblotting were performed as previously described [32 (link)]. For immunoprecipitation with anti-FLAG or anti-HA antibodies, the cell lysates were incubated with EZview red anti-FLAG M2 or anti-HA affinity resin (Sigma) for 4 or 16 hr at 4°C. After washing with lysis buffer, proteins were eluted by incubation with 1 mg/ml 3X FLAG or HA peptide for 1 hr at 4°C. For immunoprecipitation with anti-TRIM32 or anti-PB1, cell lysates were incubated with antibody and Protein A/G plus agarose (Thermo Fisher Scientific, Cambridge, MA) at 4°C for 16 hr. After washing with the lysis buffer, SDS-PAGE loading buffer was added and heated (95°C for 5 min). For immunoblotting, protein samples or 2% whole cell lysate were run on SDS-PAGE and transferred to PVDF membranes (Bio-Rad, Hercules, CA). The membranes were blocked in 5% non-fat milk in 1×Tris-buffered saline and then incubated with diluted primary antibodies at 4°C for 16 hr. Anti-rabbit or anti-mouse IgG antibodies conjugated to horseradish peroxidase (Pierce) were used as secondary antibodies. An enhanced chemiluminescence system (Pierce) was used for detection. Quantitation of immunoblots was performed using GelQuantNet software.

An in vitro protein-protein binding assay was performed as previously described [14 (link)]. pGBKT7-VP1 bait vector and pGADT7-MAT1 prey vector were used as templates to transcribe and translate in vitro, then labeled with ³⁵S-Methionine (Amersham Pharmacia Biotech) in vitro transcription-translation system (Promega), respectively, to obtain ³⁵S-labeled fusion proteins HA-MAT1 and c-Myc-VP1. The ³⁵S-Methionine-labeled HA-MAT1 and c-Myc-VP1 were incubated at room temperature, then incubated with antibody against c-Myc (Clontech) in lysis buffer, subsequently mixed with protein A/G plus-agarose (Thermo Fisher, USA) and incubated for 3 h at 4°C. The beads were washed three times with lysis buffer. The radioactive antibody–protein complexes were eluted, and subjected to SDS/PAGE and then autoradiography.