Anti spop

Cells were lysed in buffer [20 mM Tris–HCl, pH 7.3, 300 mM KCl, 0.2 mM EDTA, 0.5% Triton, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 mM glycerophosphate, and 0.1 mM Na₃VO₄, and protease inhibitors]. Total protein (50 mg) was IP with anti-SPOP (1:1000; Abcam) or unspecific mouse monoclonal IgG antibodies (I-1000; GeneTex Inc.) and 50 μl protein G-conjugated beads (Thermo Fisher). Washed beads were separated by SDS-PAGE. Following Coomassie Blue Staining, protein bands were excised and digested. After separation on a reversed phase LC column, eluted peptides were analyzed on a MALDI-QIT-TOF-based mass spectrometer with electrospray ionization (Micromass/Waters). The MS/MS data were processed using Masslynx software (Micromass), and the MASCOT (Matrix Science) search engine was used to search the NCBI non-redundant database. Protein identifications were based on a minimum random probability score of 25 and with a mass accuracy of 0.1 Da.

After 48 h transfection, cells were lysed by RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor PMSF (Wuhan Boster Biological Technology, Ltd, Wuhan, China). Protein concentration was detected with bicinchoninic acid (BCA) method. Separated proteins were denatured at 95 °C for 5 min. Equal amount of protein (20 μg) was added into each well in 12% SDSPAGE, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Afterwards, PVDF membranes were blocked in 5% skim milk for 1 h at room temperature, following the incubation with specific primary antibodies at 4 °C overnight. PVDF membranes were then sealed with secondary antibody for 1 h at 37 °C. The protein signals were visualized with ECL solution (Millipore) and scanned by QUANTITY ONE software (Bio-Rad, Hercules, CA, USA). All antibodies used in this study were listed as the following: anti-SPOP (Abcam, ab192233); anti-CHAF1A (Cell signaling Technology, CST#5480); anti-HA (Abcam, ab9110); anti-p62 (Cell signaling Technology, CST#23,214); anti-Beclin-1 (Cell signaling Technology, CST#4122); anti-β-actin (Cell signaling Technology, CST#41,470); anti-FLAG (Cell signaling Technology, CST#14,793).