Immunoprecipitation and immunoblot assays

The neural tubes in the dorsal-anterior region of the brain were isolated from E10.5 embryos in cold phosphate buffered saline (PBS) under a dissection microscope and individually collected. The neural tissues were homogenized in an immunoprecipitation (IP) lysis buffer (150 mM NaCl, 10 mM Tris-HCl; pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium ortho-vanadate, 0.2 mM PMSF, 1% Triton X-100, 0.5% Nonidet P40, protease inhibitors), or an immunoblotting lysis buffer [25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] containing protease and phosphatase inhibitors. The homogenates were centrifuged at 14,000 rpm for 15 minutes at 4 °C to obtain supernatants.

IP was performed by an initial incubation of the tissue lysates with Protein A agarose beads (Cell Signaling Technology) for 30 minutes, followed by an incubation of the supernatants with fresh Protein A agarose beads and an anti-OGT antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or IgG controls at 4 °C for 16 hours. After washing three times with the lysis buffer, precipitated proteins were eluted in Laemmli SDS buffer and subjected to immunoblot assay.

Protein samples were resolved in 10% polyacrylamide gel using electrophoresis in presence of SDS and blotted onto polyvinylidene fluoride membranes (Millipore, Billerica, MA). After blocking with 10% non-fat milk or bovine serum albumin, the membranes were incubated with primary antibodies [Chop, cleaved Caspase-3 (Cell Signaling Technology), O-GlcNAc, OGT (F-12), OGA (Santa Cruz Biotechnology), and phosphotyrosine (ThermoFisher; PY20)] for 16 hours at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 hour at room temperature. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Images were captured and density of the bands was measured using the UVP Bioimage system (UVP).

The same membranes were stripped using Restore Western Blot Stripping Buffer (Thermo Scientific) and probed again with an antibody against β-actin (Abcam) to control for equal loading of protein samples. The values of β-actin band density were used to normalize those of the corresponding bands of interest.