Fig. 3. Human SCP1 as a substrate target for O-GlcNAcylation. The immunologically defined epitope-tagged hSCP1s (Wt and D96N) were induced in NIH/3T3/hSCP1-V5 (A) and NIH/3T3/M2-hSCP1 (B) cells for the indicated periods and hSCP1 from equal amount of total cell lysate (2 mg) were immunoprecipitated with either α-Flag antibody or α-V5 epitope antibody. O-GlcNAcylated hSCP1 was detected using the α-O-GlcNAc antibody. For reprobing the blots, the blots were washed, incubated with BlotFresh Western Blot Stripping Reagent, and remonitored the input hSCP1 proteins. (B) At 72 h of the postinduction, total cell lysates (2 mg) from the noninduced and induced cells for the indicated periods were incubated with sWGA Agarose beads. The affinity-purified hSCP1 in the precipitate was resolved onto SDS-PAGE and probed with either α-DYKDDDDK or α-V5 epitope antibody.
