Fig. 4. Determination of O-GlcNAcylation site on hSCP1. (A) The Flag-tagged hSCP1 (Wt) from NIH/3T3/M2-hSCP1 cells (20 mg of total cell lysate) was subjected to the immunoprecipitation with α-Flag-Agarose. The immunoprecipitates were subjected to 6-15% SDS-PAGE and stained with CBB. Arrow indicates hSCP1. (B) A small portion of immunoprecipitate was subjected to Western blot analysis with α-DYKDDDDK antibody. (C) The Q-TOF spectrum and the sequencing results of a GlcNAc-modified peptide corresponding to residues 36-50 are shown. S⁴¹ residue in hSCP1 is an O-GlcNAcylation site. The expected increase in mass by the O-GlcNAc modification is 203.2 Da. (D) Whole cell lysates of NIH/3T3 transfected with V5-tagged hSCP1 (empty vector (pcDNA3), Wt and S⁴¹A) were immunoprecipitated with V5-epitope Agarose beads and characterized by Western blot analysis with α O-GlcNAc antibody. The protein input was monitored with α-V5 antibody after stripping the blot. Arrow head indicates IgG light chain.