Figure 4.

OGA-mBirA-HA and Myc-mBirA-OGA biotinylate proximal proteins differentially in response to oxidative stress. U2OS cells were transfected with pcDNA3.1, OGA-mBirA-HA, or Myc-mBirA-OGA and treated with biotin (25 μm, 16 h) in the presence or absence of H₂O₂ (2.5 mm, 2 h). A, equal amounts of protein (5 μg; denaturing TCL lysis) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, O-GlcNAc, OGA, HA, Myc, and actin. n = 4. B, densitometric total lane profiles for each lane from the biotin signal in A. Asterisks are used to highlight a subset of the biotinylated signals that are altered by oxidative stress. Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.