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Fig. 4 | Cell & Bioscience

Fig. 4

From: FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia

Fig. 4

Ubiquitination of SPAST is regulated by CK2-dependent phosphorylation. A Interaction of CK2α, or Ck2β and SPAST in HEK293 cells transfected with indicated plasmids. B For In vitro kinase assay, the indicated proteins were purified from E. coli and incubated with recombinant CK2 protein in the presence of [γ32P]ATP. Autoradiography shows phosphorylated GST-SPAST. C Phosphorylation of the serine/threonine to alanine mutants of SPAST was analyzed using IP-western blotting in HeLa cells. D Immunostaining of GFP and phospho-CK2 substrate in HeLa cells transfected with GFP empty or GFP-SPAST-M1 plasmids. Scale bar: 50 μm. E After transfection of Flag-SPAST-M1 or M87 plasmids in HeLa cells, phosphorylation of SPAST-M1 or M87 was analyzed by IP-western blotting from nuclear and cytoplasmic fractions of HeLa cells. F HEK293 cells were transfected with indicated plasmids and treated with CX-4945 (for 24 h with 5uM) or calyculin A (for 15 min with 10 nM), followed by isolation of nuclear and cytoplasmic fractions and analysis using western blotting. G The poly-ubiquitination of SPAST-M1 was analyzed by IP-western blotting in the HEK293 WT or CK2β-Cas9 stable cell lines. H The poly-ubiquitination of SPAST WT or phosphomimetic mutant (triple aspartic acid substitution mutations; T530D, S545D, and S547D) was analyzed by IP-western blotting in the HEK293 cells. I The protein stability of WT and phosphomimetic mutant of SPAST was analyzed by western blotting after CHX treatment. J The mRNA level of WT and phosphomimetic mutant of SPAST under the same conditions as 4I. All experiments were performed in triplicate, and results are representative of three independent experiments

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