Fig. 3.

LARGE overexpression in POMT2 KO neural stem cells did not functionally glycosylate α-DG. POMT2 KO neural stem cells were infected with Ad-LARGE and cultured for 2 days. Immunoprecipitation with VIA4-1 (A) and anti-β-DG (B) antibodies was carried out, followed by immunoblot with anti-β-DG and IIH6C4 antibodies and laminin overlay assays. (A, lane 1) VIA4-1 immunoprecipitate of wild-type cells showed immunoreactivity to anti-β-DG (asterisk) and IIH6C4 antibodies and bound to laminin. (A, lane 2) VIA4-1 immunoprecipitate of wild-type cells overexpressing LARGE showed immunoreactivity to anti-β-DG (asterisk) and dramatically increased immunoreactivity to IIH6C4 and laminin-binding activity. (A, lane 3) VIA4-1 immunoprecipitate from POMT2 KO cells showed no immunoreactivity to anti-β-DG and IIH6C4 with no laminin-binding activity. (A, lane 4) Overexpressing LARGE in POMT2 KO cells generated IIH6C4 immunoreactivity at higher molecular weight and laminin-binding activity but showed no β-DG in VIA4-1 immunoprecipitate. (B, lane 1) Anti-β-DG immunoprecipitate from wild-type cells showed IIH6C4 immunoreactivity and laminin-binding activity. (B, lane 2) Overexpressing LARGE in wild-type cells increased IIH6C4 immunoreactivity and laminin-binding activity in anti-β-DG immunoprecipitate. (B, lanes 3 and 4) IIH6C4 immunoreactivity and laminin-binding activity were not detected in anti-β-DG immunoprecipitate from POMT2 KO cells with or without LARGE overexpression. (B, lane 5) Anti-β-DG immunoprecipitate from POMGnT1 KO cells showed very weak IIH6C4 immunoreactivity at ∼70 kDa with very weak laminin-binding activity in this assay. (B, lane 6) Overexpressing LARGE in POMGnT1 KO cells increased IIH6C4 immunoreactivity and laminin binding in anti-β-DG immunoprecipitate. (B, lanes 7 and 8) As a control, anti-β-DG did not precipitate any IIH6C4 immunoreactivity and laminin-binding activity in DG KO cells with or without LARGE overexpression. No β-DG was detected in DG KO cells as expected.