Figure 3. Identification and functional analysis of nitrated tyrosine residues in FSHR protein. (A) Identification of tyrosine nitrated sites in FSHR protein. The purified FSHR proteins from KGN cells were subject to MALDI-TOF MS analysis. Peaks with m/z 1489.66, m/z 1509.64, m/z 2017.07 and m/z 2679.51 corresponding to Y322 single nitration, Y654 single nitration, Y684 double nitration and Y626 single nitration were marked respectively. (B) Membrane expressions of FLAG-WT and its mutants. Relative protein expressions of FLAG-WT, Y322A, Y626A, Y654A, Y684A and Y626F in membrane fractions of KGN cells were determined by immunoblots (NaK-ATPase as internal standard of membrane proteins). (C) Representative photo-micrographs of Alexa Flour 488 staining in KGN cells transfected with FLAG-WT, Y322A, Y626A, Y654A, Y684A and Y626F. They were examined by confocal microscopy, where Green fluorescence indicated Alexa Flour 488-positive FSHR proteins, red fluorescence indicated DiR-positive membrane, and blue fluorescence indicated Hoechst 33258-positive nuclei (Scale bar: 10 μm). (D) Relative ratios of FLAG-WT and its mutants to membrane were determined as the ratios of Alexa Flour 488 density to DiR intensity. Open triangle: p<0.05 vs. FLAG-WT (n = 3–6).