Af2330

Immunoblot analysis was performed as described previously (Kawahara et al., 2009). The blotted membraneswere incubated with one of the following antibodies: 9F5 (1 µg ml⁻¹), ED1 (1 µg ml⁻¹; MCA341GA; Serotec), rabbit anti‐Iba1 (1 µg ml⁻¹;019‐19741; Wako), goat anti‐GPNMB (1 µg ml⁻¹;AF2330; R&D Systems), rabbit anti‐furin (1:500; PA1‐062; Affinity BioReagents), goat anti‐CD40 (1:100; T‐20; Santa CruzBiotechnology), goat anti‐CD86 (1:500; 421340; Genezyme Techne), or mouse anti‐β‐actin (1:2,000; AC15; Sigma–Aldrich) antibodies.
To determine the amount of N‐linked glycosylation, the cell lysate from rat type 1 MG was first denatured in 1% sodium dodecyl sulfate (SDS) (5 min, 60°C). Deglycosylation was then performed with 60 U ml⁻¹ peptide‐N‐glycosidase F (PNGase F;Boehringer Mannheim) in phosphate buffer [0.2M Na₂HPO₄‐NaOH (pH 7.5) containing 10 mM EDTA, 0.5% NonidetP‐40, and protease inhibitors] for 24 hr at 37°C (SDS concentration during the PNGase F incubation: 0.05%). O‐Glycan chains were analyzed via digestion with α‐2,3,6,8,9‐neuraminidase (sialidase; Calbiochem) and endo‐α‐N‐acetylgalactosaminidase (O‐glycosidase; Calbiochem), according to the manufacturer's instructions. Removal of sugar chains was analyzed by using SDS‐PAGE and immunoblotting as described above.