Figure 7.

Oxidative stress induces the association of OGA with FAS, FLNA, HSC70, and OGT. U2OS cells were treated with Vehicle (V) or H₂O₂ (2.5 mm, 1–3 h). n = 3. A, anti-OGA antibody (IP: OGA; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous OGA from NETN cell lysates (500 μg), of which 1.5–2% (input) and 30–40% (immunoprecipitate) were analyzed by SDS-PAGE. OGA, FAS, FLNA, HSC70, OGT (positive control), and actin (loading/negative control) were detected by Western blotting. B, anti-FAS antibody (IP: FAS; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous FAS from NETN cell lysates (250 μg), of which 3% (input) and 60% (immunoprecipitate) were analyzed by SDS-PAGE. FAS, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. C, U2OS cells were transfected with pcDNA3.1 (control) or pCMV-SPORT6 V5-FAS (test). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (300 μg), of which 1.7% (input) and 33.3% (immunoprecipitate) were analyzed by SDS-PAGE. V5, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. A–C, FAS (CST) and FAS (NB) represent anti-FAS antibody from Cell Signaling Technology and Novus Biologicals, respectively. To ensure that images were in the linear range, Western blot exposures from the input and immunoprecipitated fractions are often different. The exposure lengths for the test and control isotype antibody immunoprecipitates are always identical. The migration of molecular mass (MW) markers is indicated.