Journal: The Journal of Clinical Investigation

Article Title: Four subtypes of disease-causing missense mutations underlie pathogenic protein interactions in neurodegenerative VPS13A disease

doi: 10.1172/JCI200890

Figure Lengend Snippet: ( A ) Structural comparison of XK and XKR2. AlphaFold 3–predicted structures of mouse XK (AlphafoldDB, Q9QXY7 ) and XKR2 (AlphafoldDB, Q5GH68 ), excluding their N- and C-terminal regions, are shown. The β-hairpin region is enclosed in a red box. ( B–E ) Functional analysis of XKR2. Xk –/– DKO -P2X7 cells were transfected with mEGFP-tagged mouse XK or XKR2. ( B ) Whole-cell lysates were separated by SDS-PAGE and immunoblotted with an anti-GFP Ab or stained with CBB. ( C ) GFP fluorescence was observed using a microscope. Scale bar: 5 �m. ( D ) Membrane fractions were separated by BN-PAGE and immunoblotted with anti-VPS13A or anti-GFP Abs or stained with colloidal gold protein stain. Red arrowhead indicates VPS13A-XK-mEGFP or XKR2-mEGFP complex; band marked with an asterisk is an unidentified protein; black arrowhead indicates uncomplexed XK or XKR2. ( E ) Cells were treated with ATP and analyzed by flow cytometry for PtdSer exposure. ( F–J ) Effect of XKR2 missense mutations on VPS13A binding and scramblase activity. Xk –/– DKO -P2X7 cells were transformed with WT, E151A, E153A, or E151A/E153A DM mouse XKR2-mEGFP. ( F ) Whole-cell lysates were separated by SDS-PAGE and immunoblotted with anti-GFP Ab. ( G–I ) Membrane fractions were separated by BN-PAGE ( G and I ) and SDS-PAGE ( H ) and immunoblotted with anti-GFP Ab ( G ) or anti-VPS13A Ab ( H and I ). Red arrowheads indicate the VPS13A-XKR2 complex; the black arrowhead indicates uncomplexed XKR2. The band marked by an asterisk is an unidentified protein. ( J ) Cells were stimulated with ATP, and PtdSer exposure was analyzed by flow cytometry using Cy5–annexin V. In ( E and J ), PtdSer exposure is expressed as MFI. Data are presented as the mean ± SD (bar) of 3 independent experiments. Statistical significance was determined using Šídák’s multiple comparison test. ** P < 0.01, **** P < 0.0001.

Article Snippet: PlasMem Bright Red ( ) was purchased from Dojindo (346-09771), ERseeing (endoplasmic reticulum green) from Funakoshi (FDV-0038) , and colloidal gold protein stain from Bio-Rad Laboratories (1706527). ( p- Amidinophenyl)methanesulfonyl fluoride hydrochloride ( p -APMSF) was obtained from Wako (019-26331).

Techniques: Comparison, Functional Assay, Transfection, SDS Page, Staining, Fluorescence, Microscopy, Membrane, Flow Cytometry, Binding Assay, Activity Assay, Transformation Assay

Journal: The Journal of Clinical Investigation

Article Title: Four subtypes of disease-causing missense mutations underlie pathogenic protein interactions in neurodegenerative VPS13A disease

doi: 10.1172/JCI200890

Figure Lengend Snippet: ( A ) Stable expression of VPS13A missense mutations. Vps13a –/– DKO- P2X7 cells were stably transformed with WT or the indicated mouse VPS13A mutants. Whole-cell lysates from DKO -P2X7, Vps13a –/– DKO -P2X7 (none), and mutant transformants were analyzed by SDS-PAGE and Western blotting using anti-VPS13A Ab. The CBB-stained membrane is shown. Red arrowhead indicates VPS13A; * indicates a nonspecific band. ( B and C ) ATP-induced PtdSer exposure. Transformants expressing WT or mutant mouse VPS13A were treated with ATP, stained with Cy5–annexin V, analyzed by flow cytometry, and expressed as MFI. Data are presented as mean ± SD (bar) of more than 3 experiments. Statistical significance was determined using Dunnett’s multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ( D ) Complex formation with XK. Membrane fractions were analyzed by BN-PAGE (upper panel) and SDS-PAGE (bottom panel) and Western blotting with anti-VPS13A or anti-XK Abs. Red arrowheads indicate the mouse VPS13A–XK complex; the black arrowhead indicates free XK. Membranes were stained with CBB ( A ) or colloidal gold protein stain ( D ). In ( A and D ), the band intensity detected by Western blotting was quantified by densitometry and expressed relative to that detected in cells expressing WT-VPS13A.

Article Snippet: PlasMem Bright Red ( ) was purchased from Dojindo (346-09771), ERseeing (endoplasmic reticulum green) from Funakoshi (FDV-0038) , and colloidal gold protein stain from Bio-Rad Laboratories (1706527). ( p- Amidinophenyl)methanesulfonyl fluoride hydrochloride ( p -APMSF) was obtained from Wako (019-26331).

Techniques: Expressing, Stable Transfection, Transformation Assay, Mutagenesis, SDS Page, Western Blot, Staining, Membrane, Flow Cytometry, Comparison