Fig 1.
Schematic diagram of the principle of WGA-SDS-PAGE.
A whole cell lysate contains proteins with different O-GlcNAcylation states ranging from no (A), through partial (B) to high (C) O-GlcNAcylation. In the WGA-copolymerized acrylamide gel layer, the migration of the O-GlcNAcylated proteins is retarded, thereby producing slower-migrating bands in the separating gel.
Fig 2.
Separation and detection of O-GlcNAcylated HA-Tab1 by WGA-SDS-PAGE.
(A) Ectopic expression of OGT augments O-GlcNAcylation of Tab1. HA-Tab1 was transiently expressed with either Flag-OGT or Myc-OGA in HEK293 cells. Immunoprecipitated (IP) HA-Tab1 was separated by standard [WGA(-)]-SDS-PAGE, and probed for O-GlcNAcylation using WGA-HRP (top panel) or the anti-O-GlcNAc RL2 Ab (2nd panel). The O-GlcNAcylation levels of proteins in the total cell lysates are also shown (4th panel). Actin was used as a loading control (bottom panel). (B) O-GlcNAcylated Tab1 forms are separated on WGA-SDS-PAGE. An aliquot of the whole cell lysates used in (A) was separated on WGA-SDS-PAGE, followed by immunoblotting with an anti-HA Ab (upper panel).
Fig 3.
The effects of WGA concentration and length of the WGA-gel on the electrophoretic-mobility of O-GlcNAcylated proteins during WGA-SDS-PAGE.
(A) An increase in WGA concentration improves the efficacy of separation of O-GlcNAcylated HA-Tab1. HA-Tab1 was co-expressed together with Flag-OGT or Myc-OGA in COS-7 cells. The cell lysates were separated by WGA-SDS-PAGE with gels containing the indicated concentrations of WGA, followed by immunoblotting with an anti-HA antibody (top and 2nd panels). The length of the WGA-gel layer was fixed at 9-mm. An aliquot of the same cell lysates was also separated with standard SDS-PAGE, and immunoblotted with an anti-HA antibody (3rd panel). The expression levels of Flag-OGT and Myc-OGA are also shown (4th and bottom panels). (B) Increase in the length of the WGA-gel layer enhances the efficacy of separation of O-GlcNAcylated Tab1 and the resolution of the protein bands. WGA-gel layers of different lengths (3, 6, and 9 mm) were prepared, and aliquots from the same cell lysate used in (A) were then separated on these WGA-SDS-PAGE gels. HA-Tab1 was detected with an anti-HA antibody as in (A). The WGA concentration was fixed at 3.75 mg/ml.
Fig 4.
Detection and separation of endogenous O-GlcNAcylated proteins by WGA-SDS-PAGE.
(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).