B26302

For worm samples, 30,000 young adult worms with the indicated genotype were washed off by M9 buffer and resuspended in 3 ml lysis buffer (50 mM tris-HCl pH 8.0, 137 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, proteinase inhibitor). Samples were homogenized with a glass homogenizer and sonicated. For HEK293T cells, cells in one 10 cm dish with 90% confluence were washed with 1X PBS buffer, then resuspended in 1 ml lysis buffer and sat on ice for 30 min. The worm or cell lysate was centrifuged at 20,000 × g for 15 min. The supernatant was then transferred into a new tube and rotated at 4 °C overnight in the presence of the designated antibody anti-GFP antibody (Abcam #ab290, 1 µl per sample), anti-FLAG magnetic beads (Sigma #M8823, 40 µl per sample), anti-Myc magnetic beads (Bimake #B26302, 20 µl per sample). For anti-GFP immunoprecipitation, protein G beads (Invitrogen #10004D, 40 µl per sample) were subsequently added to each sample and rotated at 4 °C for additional 2 h. After binding, the beads were washed three times with lysis buffer and boiled in 50 µl 2X SDS Laemmli buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol, 0.02% bromophenol blue, 0.125 M Tri-HCL, pH 6.8) at 95 °C for 10 min.

The plasmids (Myc-PTEN or Myc-DUSP7, Flag-SPOP^cyto, HA-Ub) were transiently cotransfected into 293T cells. After transfection for 24 h, 293T cells were treated with different doses of compound for 24 h. The cells were then treated with 10 μM protease inhibitor MG132 (MedChemExpress (Monmouth Junction, NJ, USA), HY-13259) for another 4 h before harvesting. Next, the cells were lysed in denaturing buffer (1% SDS, 50 mM Tris-HCl, 0.5 mM EDTA, 1 mM DTT, pH 7.5). The lysates were incubated for 5 min at 100 °C immediately, and then sonicated and diluted with cell lysis buffer for Western and IP. Approximately 80% of the total lysates were immunoprecipitated with anti-Myc-conjugated magnetic beads (Bimake, B26302) for 2 h at room temperature, and the other lysates were used as input. The magnetic beads were then washed 3 times with PBST, and the immunoprecipitated proteins were eluted with 1 × SDS loading buffer at 100 °C for 5 min. The ubiquitination levels were detected using a Western Blot assay.