Fig. 3

The SCF^FBXL17 E3 ligase complex induces poly-ubiquitination and proteasomal degradation of SPAST. A HEK293 cells were transfected with indicated plasmids followed by 10 μM MG132 treatment for 16 h and analyzed using western blotting. B After transduction on ReNcell CX with a lentiviral-vector expressing FBXL17, protein expression was detected using western blotting in the presence or absence with MG132. C HEK293 cells were transfected with HA-tagged SPAST-M1 and Flag-tagged FBXL17 plasmids followed by 50 μg/ml cycloheximide (CHX) treatment for indicated times, analyzed using western blotting, and protein intensities quantified using ImageJ software. The assays were performed in triplicates and error bar represents SD. (*P < 0.05; ** P < 0.01). D HEK293 cells were transfected with indicated plasmids followed by MG132, and total cell lysates were separated into nuclear and cytoplasmic fractions. The same amounts of cell lysates were immunoprecipitated with anti-Flag-magnetic bead followed by western blotting with indicated antibodies. E HEK293 cells were transfected with Flag-SPAST-M1 or M87 plasmids, treated with MG132, and separated into subcellular fractions. Poly-ubiquitination of SPAST-M1 or M87 was analyzed using immunoprecipitation and western blotting. F In vitro ubiquitination assays were performed by incubating indicated proteins with the ATP-regeneration buffer system at 37 ℃ for 1.5 h. Proteins were purified from the E. coli expression system. The cytosolic S100 extract was prepared from HeLa cells transduced with Lenti-FBXL17 shRNA for 72 h. G In vitro ubiquitination assays were performed with GST-SPAST-M1-WT or K554R protein under the same conditions in (F), analyzed by western blotting. All IP and WB experiments were performed in triplicate, and results are representative of three independent experiments