(a) Western blotting analysis of the mGluR3 ECD proteins. The samples were loaded in the presence (lanes 1 and 2) and absence (lanes 3 and 4) of DTT. The positions of the molecular-weight markers are shown on the left (kDa). Lanes 1 and 3, mGluR3 ECD wild type with a His-tag; lanes 2 and 4, N414Q mutant with a His-tag. (b) SDS–PAGE analysis of the wild-type and mutant LBDs. These proteins were analyzed under reduced conditions. The gel was stained with silver nitrate. Lane 1, wild type; lane 2, N209Q; lane 3, N292Q; lane 4, N414Q; lane 5, N439Q. (c) The site-specifically unglycosylated mutant proteins of ECD without a His-tag were analyzed by SDS–PAGE. The purified samples were loaded in the presence (lanes 1–3) and absence (lanes 4–6) of DTT. The gel was stained with Coomassie Brilliant Blue. The positions of the molecular-weight markers are shown on the left (kDa). Lanes 1 and 4, N414Q; lanes 2 and 5, N292/414Q; lanes 3 and 6, N414/439Q.