Polyclonal antibodies targeting ogt

For immunoprecipitation, aliquots of cell homogenates were incubated with polyclonal antibodies targeting OGT (4 μg/ml) (Abcam, Cambridge, UK) overnight at 4 °C. Protein G-Sepharose beads (GE Healthcare, Chicago, IL, USA) were added to the supernatant and incubated for 3 h. The beads were washed thoroughly with lysis buffer and then eluted by boiling for 3 min in SDS-PAGE sample buffer (Beyotime).
eNOS Pull-down Assay
In BAEC and rat thoracic aorta, eNOS was extracted from the lysate by a nity precipitation using 2′,5′-ADP Sepharose beads (GE Healthcare). Cell/tissue lysates were and mixed with prepared 2′,5′-ADP Sepharose resins (50% slurry) at 4 °C for 2 h with gentle shaking. The mixture was then centrifuged at 13800 × g for 1 min. The supernatant was discarded, and the resins were washed with washing buffer (PBS supplemented with 500 mM NaCl) three times. Bound proteins were eluted by boiling the resins in 50 μl SDS-PAGE sample buffer for 10 min. For transfected cells, eNOS was extracted by a nity precipitation after transfection using the His-tag protein puri cation Kit (Beyotime) per the manufacturer's protocol.