Purification and Analysis of Ubiquitylated Proteins

When indicated, ubiquitylated proteins were purified by histidine (His) pull-down. To this end, cells expressing His-tagged ubiquitin were lysed in denaturing buffer, containing 8 m urea, 100 mm Na₂PO₄, 10 mm Tris-HCl, pH 8.0. Lysates were applied to cobalt resin (Thermo Fisher Scientific) for 2 h, and then beads were washed twice with buffer containing 8 m urea, 100 mm Na₂PO₄, 10 mm Tris-HCl, pH 6.3; denatured in NuPAGE sample buffer; and loaded on a 6% polyacrylamide gel.
GST and GST-Pdcd4 recombinant proteins were produced in Escherichia coli BL21, as described previously (34 (link)). For GST pull-down, HEK293T cells (3 × 10⁶) were transfected with pCMV6-IBtkα-FLAG or IBtkα-FLAG mutant plasmids (4 μg), and 48 h later, cells were lysed in RIPA buffer. The lysate (1 mg) was incubated for 1 h with GST or GST-Pdcd4-WT and mutants at 4 °C under constant agitation. Then glutathione-Sepharose beads (30 μl) (GE Healthcare) were added to the mix for a 1-h incubation at 4 °C. Subsequently, beads were washed three times with RIPA buffer, and the bound proteins were eluted from beads by boiling in SDS sample buffer. Protein samples were subjected to electrophoresis on NuPAGE 4–12% polyacrylamide gel and analyzed by immunoblotting with the appropriate antibodies.