Ripa buffer

Cultured macrophages or livers of the mice were collected and lysed by RIPA buffer (Beyotime, Shanghai, China) on ice for 20 min, and liver tissue was homogenized with RIPA buffer on ice, followed by centrifugation at 12,000× g, 4 °C for 20 min. The protein concentration was measured by a commercial BCA kit (Beyotime Biotechnology, Shanghai, China). Approximately 30 mg of total protein was separated by 12% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk powder at room temperature for 2 h, then overnight at 4 °C with primary antibodies targeting phospho-JNK (Cell Signaling Technology, Danvers, MA, USA; 1:1000 dilution), phospho-ERK (Cell Signaling Technology; 1:1000 dilution), phospho-p38 (Cell Signaling Technology; 1:1000 dilution), and tubulin (Beyotime, Shanghai, China; 1:1000 dilution) as a loading control. Then, the membranes were washed with washing buffer three times and incubated with HRP-conjugated goat anti-rabbit or mouse IgG secondary antibody (proteintech, Wuhan, China; 1:10,000 dilution) for 2 h at room temperature and washed with washing buffer three times. Detection was performed using an enhanced chemiluminescence reagent (Biosharp, Hefei, China). Images were acquired using a Bio-Rad chemiluminescence imaging system and were analyzed with Image J software.

Cells were lysed in RIPA buffer (Beyotime, P0013J) supplemented with 1 × proteinase inhibitor (Beyotime, P1006). Immunoprecipitation with 2.5 μg of RPA32 antibody or 2.5 μg of isotype IgG (Sigma, I5006) per sample was performed using Protein G Dynabeads (Thermo, 88847) according to the manufacturers’ protocol.
For western blot, total protein was extracted using RIPA buffer (Beyotime, P0013J). Proteins were subjected to 4%-12% SDS-PAGE gels for separation and transferred to polyvinylidene fluoride (PVDF) membrane (Roche, 03010040001). The membrane was blocked with 5% non-fat milk at room temperature for 2 h and then incubated with primary antibodies at 4 °C overnight. After incubation with anti-mouse or rabbit HRP-conjugated secondary antibodies for 1 h at room temperature, the bands were revealed using ECL reagent (Beyotime, P0018FS). Antibodies used were listed in Supplementary Table 2.

PAX5 together with HA-Ub and NTRK2-243aa plasmids were transfected into HEK-293T and incubated with 10 μM MG132 for 4 h. Cell lysates were prepared using the RIPA buffer (Beyotime Institute of Biotechnology, Jiangsu, China) mixed with 10 mM N-ethylmaleimide and protease inhibitors (Beyotime Institute of Biotechnology, Jiangsu, China). After 30 s of ultrasonic treatment, the supernatant was boiled at 95 °C for 15 min, followed by dilution with RIPA buffer containing 0.1% SDS (Beyotime Institute of Biotechnology, Jiangsu, China), and then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was incubated with anti-PAX5 antibody and 30 μL protein A-Sepharose beads (Sigma-Aldrich, MO, USA) overnight at 4 °C. After extensive washing and centrifugation, bound proteins were washed out by boiling with 2 × SDS sample loading buffer at 95 °C for 5 min, and PAX5 ubiquitination was determined by western blot analysis. The primary antibodies are shown in Supplementary Table 4.