Akama et al. 10.1073/pnas.0603248103. |
Supporting Table 1
Supporting Table 2
Supporting Figure 7
Supporting Text
Fig. 7. Electron micrographs of kidney, heart, small intestine, and pancreas from wild-type and MII/MX double-null animals. Note the presence of large vacuoles and enlarged mitochondria in cells from the double-null kidney, whereas these abnormalities are not seen in heart, small intestine, and pancreas.
Supporting Text
Materials and Methods
Histological analysis and electron microscopy. For histological analysis, embryos and tissues were fixed in 4% paraformaldehyde in PBS at 4°C for 20 h and embedded in a paraffin block. Tissue sections were either stained with hematoxylin/eosin or subjected to apoptosis analysis using the ApopTag kit (Chemicon, Temecula, CA). For electron microscopy, fresh tissue samples obtained from wild-type and mutant mice were immediately fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 20 h at 4°C. After washing with the same buffer, tissues were fixed further with 1% OsO4 at 4°C for 90 min. Fixed tissues were dehydrated through graded alcohols and embedded in Quetol 812 (Nissin EM, Tokyo). Semithin sections prepared from tissue blocks were stained with toluidine blue. Ultrathin sections of 70-nm thickness were then prepared from trimmed blocks and collected on single-slot copper grids. Grids were dried and stained with uranyl acetate and lead citrate. Ultrathin sections were inspected by using a transmission electron microscope JEM1230 (JEOL) at 80 kV accelerating voltage.
Lectin blot and lectin histochemistry.
Whole embryonic day (E)15 mouse embryos were homogenized individually in 800 ml of TKS buffer (50 mM Tris/HCl, pH 7.5/25 mM KCl/1 mM PMSF/0.25 M sucrose) plus 4 ml of Proteinase Inhibitor Mixture (Sigma) on ice, and the homogenate was centrifuged at 600 × g for 10 min at 4°C. The supernatant was again centrifuged at 200,000 × g for 30 min at 4°C, and the resulting precipitate was dissolved in 1% SDS and used as the membrane fraction. After determining protein concentration, this fraction was subjected to SDS/PAGE, and the proteins were transferred to a poly(vinylidene difluoride) (PVDF) filter. The filter was blocked with 5% BSA-PBS including 20 mg/ml avidin for 20 h at 4°C. After washing three times with PBS including 0.05% Tween-20 (PBST), the filter was reacted with 2.5 mg/ml biotinylated lectin (Vector Laboratories, Burlingame, CA) in 5% BSA-PBS including 10 mM biotin for 1 h at room temperature. The filter was washed three times with PBST and reacted with horseradish peroxidase (HRP)-avidin for 30 min. The filter was washed three times with PBST, and bound signals were visualized by using a SuperSignal West Pico Kit (Pierce Biotech). For lectin histochemistry, thin- sections from a paraffin-embedded E15 embryo were subjected to the lectin analysis described above, except diaminobenzidine (DAB) was used for signal detection. For lectin cytochemistry, primary-cultured fibroblasts prepared from an E15 embryo and cultured in DMEM/10% FBS were washed once with PBS and fixed with 4% paraformaldehyde-PBS for 15 min on ice. Cells were blocked and reacted with biotinylated E-phytohemagglutinin (PHA) lectin as described above, and signals were visualized by reacting with FITC-labeled avidin and detected under a fluorescence microscope.Recombinant protein expression in cultured cells.
An expression vector containing full-length coding sequence of either a-mannosidase II (MII) or a-mannosidase IIx (MX) was transfected into mouse fibroblasts prepared from an E15 MII/MX double-null embryo using LipofectAMINE plus reagent (Invitrogen) as recommended by the manufacturer. Transfected cells were cultured for 2 days and subjected to lectin cytochemistry as described above. To prepare soluble enzymes, an expression vector encoding each enzyme was transfected into COS-1 cells using LipofectAMINE plus reagent, and cells were cultured for 2 days. Culture medium of transfected cells was replaced with Opti-MEM (Invitrogen), and cells were further cultured for 1 day. The medium was recovered and concentrated by a Centriprep-30 filter unit, (Millipore), and an equal volume of glycerol was added before storage at –20°C until use.