Ctd110

The presence of O-GlcNAc on proteins was detected by Western blotting using a pan-GlcNAc mouse monoclonal antibody (CTD 110.6, Covance). For characterization of cytoplasmic and membrane fractions, we used a mouse monoclonal anti–β-actin antibody (ab8224, Abcam), a rabbit polyclonal anti-calnexin antibody (sc-11397, Santa Cruz Biotechnology), and a mouse monoclonal anti-Na(+)/K(+) adenosine triphosphatase α-1 subunit antibody (a6F, Developmental Studies Hybridoma Bank, Iowa City, IA, USA). Briefly, the membrane was blocked for 1 hour using 3% BSA or 5% milk diluted in tris-buffered saline with Tween 20 (TBST) (0.1% Tween) while shaking. Antibodies, diluted 1:1000 in 3% BSA or 5% milk in TBST, were added to the membrane overnight, followed by several washes with TBST. Next, the membrane was incubated with appropriate horseradish peroxidase (HRP)–conjugated secondary antibody for 1 hour at room temperature while shaking. Blots were developed after thorough TBST washes using an enhanced chemiluminescence system (EZ-ECL, Biological Industries) according to the manufacturer’s manual.

O-GlcNAcylated proteins in metaphase II (MII) oocytes and preimplantation diploids from the 2-cell to blastocyst stage were detected by immunostaining with two different antibodies. CTD110.6 (Covance, Princeton, NJ, USA) and RL2 (Novus Biological, Littleton, CO, USA) are primed to synthetic peptides containing serine-O-GlcNAc, and cytoplasmic and intranuclear O-linked glycoproteins, respectively. When samples were treated with CTD110.6, oocytes and diploids were fixed with 4% PFA supplemented PBS-PVA at 4 C overnight and then permeabilized by PBS-PVA containing 0.2% Triton X-100 for 20 min. For the RL2 treatment, samples were fixed and permeabilized as described for OGA detection. Fixed oocytes and diploids were washed twice with PBS-PVA and stored at 4 C in BPP until use. Immunostaining was carried out as described above for OGA detection, except that CTD110.6 (1:100) or RL2 (1:100) was used as primary antibody. Alexa Fluor 546 labeled anti-mouse IgM and Alexa Fluor 488
labeled anti-mouse IgG (Life Technologies) were used as secondary antibodies for CTD110.6 and RL2, respectively. Mounting and observation were carried out as described above for OGA detection.