Anti myc agarose beads

The supernatant was obtained by lysing the cells according to the IB protocol as previously described. For endogenous IP assay, cell extracts were pre-cleared with Protein A/G beads (80105 G, ThermoFisher Scientific) and control IgG, mixed and incubated at 4 °C for 1 hour. After centrifugation, 0.5 ml of supernatant was added with 3 μg of antibody for protein pull-down. An additional 0.5 ml of supernatant was supplemented with an equivalent amount of homologous IgG antibody as a control and incubated overnight at 4 °C. The following day, 50 μl of Protein A/G beads (50%) were added to each tube and pulled down for 3 hours at 4 °C. Magnetic beads were washed three times with Tris-buffered saline (TBS, R017R.0000, ThermoFisher Scientific) containing 0.05% Tween-20 (85113, ThermoFisher Scientific) for 5 minutes each. Bound proteins were washed with 2× Laemmli buffer (S3401-10VL, Sigma-Aldrich) by centrifugation at room temperature for 10 minutes. For IP of overexpressed tagged fusion proteins, cell extracts were pre-cleared with Protein A/G beads and then incubated overnight at 4 °C with either anti-MYC agarose beads (20168, ThermoFisher Scientific) or anti-HA magnetic beads (88836. ThermoFisher Scientific). IB analysis was performed using antibodies against HA or Myc.

The in vitro deubiquitination was performed as described (55 (link)). SFB-SPRTN alone or HA-Ubiquitin and SFB-SPRTN together were expressed in HEK 293T cells. At 24 h posttransfection, cells were lysed in NETN buffer. SFB-tagged ubiquitinated SPRTN was purified by immunoprecipitation with streptavidin-sepharose beads followed by elution with 2 mM biotin on an end-to-end rocker at 4 °C for 1 h. The eluate for SFB-HA-Ub-SPRTN was then immunoprecipitated with anti-HA-beads (Thermo Fisher Scientific; Cat#88836) and eluted with 2 mg/ml HA peptide (Sigma-Aldrich; Cat#I2149) at 37 °C for 10 min. In a parallel experiment, Myc-USP11 and Myc-USP11 C318S were expressed in HEK 293T cells for 24 h. Myc-USP11 or Myc-USP11 C318S was purified by immunoprecipitation with anti-Myc-agarose beads (Thermo Fisher Scientific; Cat#20168) followed by elution with 100 μg/ml c-Myc peptide (Sigma-Aldrich; Cat#M2435) on an end-to-end rocker at room temperature for 1 h. For in vitro deubiquitination assay, purified SFB-SPRTN or SFB-HA-Ub-SPRTN was incubated with purified Myc-USP11 or Myc-USP11 C318S in a deubiquitination reaction buffer (50 mM HEPES, pH 7.5, 100 mM NaCl, 5% glycerol, 5 mM MgCl₂, 1 mM ATP, and 1 mM DTT) at 30 °C overnight. The reaction mixture was terminated using SDS-PAGE buffer and analyzed by western blotting.

Transformants were cultured in liquid V8 medium for 3 d then total proteins were extracted using the BestBio Thick-wall microbial protein extraction kit. N-ethyl maleimide (NEM) was added when detecting PsATG8²² . The resulting protein extract was incubated with 25 μL (0.25 mg) anti-Flag, anti-HA, or anti-Myc agarose beads (Thermo Fisher Scientific) for 3 h at 4 °C. The combined agarose beads were then collected by centrifugation and washed three times with pre-cooled wash buffer (50 mM Tris.HCl, 0.15 M NaCl, pH = 7.4). The immunoprecipitates were separated by SDS-PAGE and detected using the corresponding antibodies.