Additional file 1 of FBXL17/spastin axis as a novel therapeutic target of hereditary spastic paraplegia
Creators: name: Kang, Hyun Mi
name: Kim, Dae Hun
name: Kim, Mijin
name: Min, Yoohong
name: Jeong, Bohyeon
name: Noh, Kyung Hee
name: Lee, Da Yong
name: Cho, Hyun-Soo
name: Kim, Nam-Soon
name: Jung, Cho-Rok
name: Lim, Jung Hwa
Publisher: name: figshare
Year: 2022
Subjects: subject: Biophysics
subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Neuroscience
subject: Pharmacology
subject: Biotechnology
subject: Developmental Biology
subject: Cancer
subject: 111714 Mental Health
subject: FOS: Health sciences
subject: Hematology

description: Additional file 1: Figure S1. (A) Schematic image of the E3 ubiquitin ligase candidates of SPAST. (B) After transfection with the Flag-SPAST-M1 construct, HEK293 cells were immunoprecipitated, and analyzed using western blotting. Figure S2. The RT-PCR band density in Fig. 1D is generated using ImageJ software. Figure S3. (A) Schematic representation of SPAST N-terminal deletion mutants. Interaction capacity between SPAST deletion mutants and FBXL17 is indicated by an asterisk. (B) BTB domain sequences of the identified BTB proteins and SPAST-M1 were represented by multiple sequence alignments (C) Schematic representation of FBXL17 N-terminal deletion mutants. Interaction capacity between FBXL17 mutants and SPAST is indicated with the symbol. Figure S4. (A) mRNA expression was analyzed under the conditions shown in Fig. 3A and quantified using ImageJ software. (B) mRNA expression was analyzed under the conditions shown in Fig. 3C. Data are mean ± standard deviation. (ns, not significant). Figure S5. (A) Multiple sequence alignment of SPAST proteins in three species. The nine lysine residues or the predicted phosphorylation sites of SPAST confirmed its interspecies functional conservation using multiple alignment software (lysine residues, marked in red boxes; predicted S/T residues, marked in blue boxes). (B) For in vivo ubiquitination assay, HEK293 cells were transfected with indicated plasmids followed by MG132 treatment, and total cell lysates immunoprecipitated with anti-Flag-agarose gel followed by western blotting with indicated antibodies. Figure S6. (A) SDS-PAGE analysis of purified His-E1, His-UbcH10b, His-FBXL17-ΔNT1 (317-717a.a.), GST-SPAST-M1, GST-KLHL, and GST. All human recombinant proteins were expressed in E. coli and purified as described in the Materials and Methods section. Purified proteins were separated using SDS-PAGE gel and visualized using Coomassie Blue staining. (B) The E1/E2 thioester assay with His-E1, His-UbcH10b, and Flag-ubiquitin. (C) HeLa cells were transduced with scrambled shRNA control lentivirus or shRNA lentivirus targeted against FBXL17. After incubation for 72 h, S-100 cytosolic extract was prepared from the cells and analyzed via western blotting using antibodies against SCFFBXL17 components. (D) SDS-PAGE analysis of purified WT or K554R of GST-SPAST-M1 and GST proteins from the E.coli recombinant protein expression system. Figure S7. (A) In vitro CK2 kinase assay against SPAST. Proteins were separated using SDS-PAGE after the in vitro kinase assay in the presence or absence of CK2 and stained with Coomassie Blue staining demonstrating equal loading of the substrate. (B) mRNA level of CK2α and CK2β was analyzed by real-time qPCR in HEK293 WT or CK2β-Cas9 stable cell line (left). Data are represented as mean + /– standard deviation calculated from three replicates. The PCR products were visualized by 2% agarose gel electrophoresis (right). (**p &lt; 0.01; ns, not significant). Figure S8. (A) mRNA expression was analyzed and quantified using RT-PCR under the conditions shown in Fig. 5B. (B) mRNA expression was analyzed using RT-PCR under the conditions shown in Fig. 5C and quantified. Data are mean ± standard deviation. (*p &lt; 0.05; **p &lt; 0.01; ns, not significant). Figure S9. (A) Rencell CX cells were treated with 0.2, 0.5 μM MLN4924 for 2 d, and analyzed using western blotting with the indicated antibodies. (B) After transfection with pEGFP plasmid, the cells were differentiated for 2 d in the presence or absence of 0.5 μM MLN4924 and stained with GFP and acetylated α-tubulin antibodies. Fluorescence intensities of acetylated α-tubulin quantified with ImageJ software. Data are mean ± standard deviation calculated from three replicates. Scale bar: 20 μm (*p &lt; 0.05). Figure S10. (A) Immunostaining of GFP and Acetylated α-tubulin in HeLa cells transfected with GFP empty or GFP-SPAST-M1 plasmids, and (B) fluorescence intensities of acetylated α-tubulin quantified using ImageJ software. Data are mean ±