Protease inhibitor mixture

For nuclear protein isolation, cells were scraped into ice-cold PBS. After centrifugation, the collected cell pellet was then resuspended in lysis buffer containing 10 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.1% Nonidet P-40, and a protease inhibitor mixture (Sigma-Aldrich); then, the cells were incubated for 10 m on ice and centrifuged. The supernatant (the cytosolic fraction) was collected, and the pellet was resuspended in buffer containing 20 mM HEPES, 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 25% glycerol, and a protease inhibitor mixture (Sigma-Aldrich). After incubating for 10 m on ice, the samples were centrifuged, and the supernatants (nuclear proteins) were collected and stored at −80 °C until analysis.

Total proteins were extracted from six-week-old rice seedlings in RIPA buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 10 mM MgCl₂, and 1 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture [Sigma, St. Louis, MO, USA]), and the supernatant was collected. The total protein concentration was determined using a Bradford protein assay kit (Bio-Rad). Proteins were detected using anti-FLAG [Sigma, St. Louis, MO, USA], anti-HDR1, anti-HD1 or anti-OsK4 [kindly supplied by Dr. Liu, BIG, CAS] antibodies at 1:4000 dilution and visualized with an enhanced chemiluminescence (ECL) reagent (GE Healthcare).
The HIS-HDR1 fusion protein were overexpressed in Escherichia coli (strain BL21) and purified to generate multi-clone antibodies. Frozen cells were extracted with 100 μl of extraction buffer (50 mM Tris-HCl. pH 7.6, 15 mM MgCl₂, 0.1 M KCl, 0.25 M sucrose, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor mixture [Sigma, St. Louis, MO, USA], and 14 mM β-mercaptoethanol). After centrifugation of the sample at 15,000 x g for 10 min, the supernatant was sampled, and the protein concentration was measured. The amount of protein form each extract was measured according to the manufacturer’s instructions [Laboratories Inc., Mountain View, CA, USA].

The cells were lysed in ice-cold RIPA lysis buffer containing a protease inhibitor mixture (Sigma) for 30 min at 4 °C. The lysates were subjected to Western blot. For co-immunoprecipitation, the 293 T cells were cultured in a 60-mm-diameter plate and transfected with various indicated plasmids. Forty-eight hours later, the cells were collected and disrupted using a lysis buffer containing a protease inhibitor mixture (Sigma) and PMSF for 30 min at 4 °C. The cell lysates were separated by centrifugation at 12,000 rpm for 15 min at 4 °C. Anti-HA agarose beads (Sigma) or Anti-FLAG agarose beads (Sigma) were mixed with the cell lysates and incubated at 4 °C for 4 h or overnight. If immunoprecipitated endogenous USP10, the rabbit anti-USP10 (Abcam) or rabbit IgG and Protein G agarose beads were used. The beads were subsequently washed four times with the cold lysis buffer and diluted with a gel loading buffer. The immunoprecipitated samples were analyzed by SDS-PAGE, followed by western blotting. Bands were immunoblotted with indicated antibodies and IRDye secondary antibodies (LI-COR) and visualized with the Odyssey infrared imaging system (LI-COR).