Fig. 2
From: FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia
SPAST interacted with FBXL17, a substrate recognition component of SCF E3 ubiquitin ligase. A Immunostaining of GFP and ER in HeLa cells transfected with GFP empty, GFP-SPAST-M1, or M87 plasmids after MG132 treatment. DAPI was used for nuclear staining. Scale bar: 20 μm. B Immunostaining of GFP and endogenous FBXL17 in HeLa cells under the same conditions in (A) C Immunoprecipitation and western blot analysis of the interaction between Flag-tagged SPAST and endogenous SCFFBXL17 complex in HEK293 cells. D Interaction of endogenous SPAST and FBXL17 was analyzed by immunoprecipitation and western blotting from ReNcell CX. E Immunoprecipitation was performed using the anti-Flag magnetic bead in HEK293 cells transfected with the indicated plasmids, and IP samples were analyzed using western blotting. F The pull-down assay with glutathione agarose beads was performed in HEK293 cells transfected with the indicated plasmids and analyzed using western blotting. G Schematic showing the F-box domain (pink) and leucine-rich repeats domain (LRRs, purple) of the FBXL family members. H Immunoprecipitation and western blot analysis from HEK293 cells transfected with the indicated plasmids. I In vitro binding assay was performed with purified GST-tagged SPAST-M1, GST-KLHL, GST-alone, and His-tagged FBXL17-ΔNT1 (318–701 a.a.) from the E.coli expression system and analyzed using western blotting. All IP and WB experiments were performed in triplicate