Detection and separation of endogenous O-GlcNAcylated proteins by WGA-SDS-PAGE.

<p>(A and B) Whole cell lysates from HEK293 cells transfected with siRNA targeting OGT or control siRNA, and treated with or without 10 μM Thiamet-G (TMG) for 24 hours were separated on WGA-SDS-PAGE (A) or standard SDS-PAGE (B). WGA-gel layer conditions were fixed at 9-mm in length and a concentration of 3.75 mg/ml. Immunoblotting was performed with an anti-AGFG1 antibody (upper panels) or an anti-Nup62 antibody (bottom panels). The black and white arrowheads indicate the O-GlcNAcylated and non-O-GlcNAcylated protein bands, respectively. Individual migration bands of AGFG1 (A, upper panel, no. 1–7) were quantified by densitometry, and the relative abundance ratio is shown in a small graph. The area of the WGA-gel layer transferred to the nitrocellulose membrane is indicated by a black bracket. Actin was used as a loading control (bottom panel, B).