Fig. 5
From: FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia
Inhibition of SCF.FBXL17 E3 ubiquitin ligase by MLN4924 stabilizes SPAST protein. A Schematic of mechanism for MLN4924-mediated inhibition of SCF complex. B The protein level of HA-SPAST-M1 in HEK293 cells treated with MLN4924 for 24 h in the presence or absence of FBXL17 expression. SPAST intensity bands were quantified using ImageJ software. C The half-life of HA-SPAST-M1 protein in HEK293 cells treated with 50 μg/ml CHX in the presence or absence of FBXL17 expression and/or MNL4924 treatment. SPAST intensity bands were quantified using ImageJ software, and the assays were performed in triplicates and error bar represents SD. D The poly-ubiquitination of HA-SPAST-M1 in HEK293 cells treated with MLN4924 at the indicated concentrations for 24 h. E ReNcell CX were transfected with a GFP-expressing vector, and incubated under neural differentiation condition for 2 d in the presence or absence of paclitaxel and MLN4924 singly and together. The cells were stained with GFP, and acetylated a-tubulin antibodies and quantified using ImageJ software. Scale bar: 20 μm. F Under the same condition as in Fig. 5E, protein expression of SPAST-M1, FBXL17, and Cullin1. G After transduction on ReNcell CX with a LV expressing shRNA for silencing of FBXL17, cells were analyzed using western blotting in the presence or absence with MG132. H and I ReNcell CX were transduced with lentiviral expressing FBXL17 shRNA (LV-GFP-shFBXL17) or scrambled non-targeting shRNA (LV-GFP-shCtrl), incubated under neuronal differentiation condition for 2 d, and immunostained with GFP, acetylated a-tubulin (as a marker for microtubule density), and Tau (as a marker for axonal length) antibodies. Scale bar: 20 μm. J Immunostaining was quantified using ImageJ software. All experiments were performed in duplicate, and results are representative of two independent experiments. (** P < 0.01)