After washing, detection of antibody binding was carried out with ECL (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA ( heterodimer) deficiency, caused by mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB -subunit to that in the native HexA -subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses. with a histidine-tag (10xHis) and the signal sequence of human lysosomal -galactosidase A (and pCXN2-vectors [10]. Then each vector was used to transform MAX Efficiency DH5 Competent Cells (Life Technologies, Carlsbad, CA, USA). Plasmid DNA-Lipofectamine 2000 (Life technologies) complexes were transfected into CHO cells according to the manufacturer’s instructions. Drug-resistant cell lines were established by double selection with hygromycin (Wako, Osaka, Japan) and G418 (Sigma-Aldrich). The CHO cell line stably expressing GM2A was cultured in EX-CELL ACF CHO medium (Sigma-Aldrich). The conditioned medium (CM) derived from each cell line was collected. Immunoblotting for the expressed GM2A was performed with anti-hGM2A polyclonal antibodies (HPA008063, Sigma-Aldrich). Briefly, aliquots of GM2A fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% (w/v) acrylamide gels. The proteins were transferred to Immobilon?-P PVDF membranes (Merck, Darmstadt, HE, Germany). After blocking with 50% (v/v) PROTAC FLT-3 degrader 1 Blocking One (Nacalai Tesque, Kyoto, Japan) in TBS [25?mM Tris (Sigma-Aldrich), 137?mM NaCl, 2.7?mM KCl, pH 7.4] at room temperature (rt) for 1?h, each membrane was treated with anti-hGM2A antibodies diluted PROTAC FLT-3 degrader 1 with Blocking One/TBS (1:1,000 dilution) overnight at 4?C. After washing with TBS containing 0.1% (v/v) Tween 20 (Sigma-Aldrich), the PROTAC FLT-3 degrader 1 membrane was treated with horseradish peroxidase (HRP)-linked anti-rabbit IgG antibodies (#7074, Cell Signaling Technology, Danvers, MA, USA, 1:1000 dilution) at rt for 1?h. After washing, detection of antibody binding was carried out with ECL (PerkinElmer, Waltham, MA, USA) according to the manufacturer’s instructions. The protein levels were determined by the test. We used the 2-tailed unpaired gene containing a 10xHis-tag and the signal sequence by utilizing plasmid vectors containing different drug-resistance genes (Fig. 1(A)), and established CHO cell lines stably expressing by sequential selection with hygromycin and G418. Although the recombinant GM2A-His proteins were secreted from the CHO cells, marked increases in the GM2A level was observed on immunoblotting with anti-GM2A antibodies in serum-free CM derived from the CHO cell line repeatedly transfected with the expression vectors. The secreted GM2A-immunoreactivity in serum-free CM of the CHO cell line transfected with two different vectors (Fig. 1B, C, lane 2V) was about two times higher than that for Agt the cell line transfected once (Fig. 1B, C, lane 1V). The expressed GM2A migrated to the 23?kDa position. After digestion with PNGase F, non-glycosylated GM2A gave a 20?kDa band, indicated the GM2A contains (sigC): without a signal PROTAC FLT-3 degrader 1 sequence. (B) Immunoblotting of CM with anti-hGM2A antibodies. Each lane contained 20 L of CM. (C) GM2A-immunoreactivity indicated as relative signal intensity of GM2A. 1 V: one vector, 2 V: two vectors transfected. (D) Immunoblotting of N-glycosylated GM2A and the digested product with PNGase F. Each lane contained 50?L of CM. (E) Purification of GM2A by Ni-column chromatography. Each fraction was separated by SDS-PAGE, and then silver staining was performed. Each lane contained 5?g of protein. CM: conditioned medium. Elu: eluted fraction. 3.2. GM2A replacement and GM2 reduction in patient fibroblasts We evaluated the GM2A function in the culture system. Cultured fibroblasts derived from a variant AB patient were treated with recombinant GM2A, and then examined by immunoblotting with anti-GM2A antibodies and immunostaining with anti-GM2 antibodies. Significant restoration of the GM2A-immunoreactivity in a cell extract was observed after treatment PROTAC FLT-3 degrader 1 with recombinant GM2A (Fig. 2A and B). We detected excessive accumulation of GM2 in lysosomes as punctate fluorescence, co-localized with LAMP-1, in untreated variant AB fibroblasts. After treatment with GM2A, the punctate fluorescence due to GM2 was markedly reduced (Fig. 2C). Open in a separate window Fig. 2 GM2A replacement.