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. Author manuscript; available in PMC: 2023 Jun 16.
Published in final edited form as: Cell Chem Biol. 2022 Feb 10;29(6):970–984.e6. doi: 10.1016/j.chembiol.2022.01.007

Figure 2. Phosphorylation of tyrosine 267 is critical for GMPR activity.

Figure 2.

A-B. SK-Mel-103 cells transduced with empty lentivirus vector (V) or vectors expressing cDNA encoding the indicated GMPR mutants were probed in immunoblotting with the indicated antibodies (top) or in Boyden’s chamber invasion assay (bottom, average +/− SEM of at least two biological replicas, Student’s t-test). C. SK-Mel-103 cells stably expressing the indicated flag-tagged proteins were used for immunoprecipitation with anti-FLAG antibodies followed by an in vitro GMPR activity assay measuring the rate of NADPH oxidation during GMPR-mediated conversion of GMP to IMP (see STAR Methods). Representative reaction progress curves are shown. The average specific activity of at least three determinations were 0.82 x 10−3 s−1 and 5.1 x 10−3 s−1 for GMPRY267F and GMPRWT, respectively (Student’s t-test; p = 0.002). D. SK-Mel-103 cells transduced with the indicated constructs or treated with mycophenolic acid (MPA, 1 μM for 24 hours) were probed in mass spectrometry to determine GTP content (average +/− SEM of ≥ 2 biological replicas, Student’s t-test). All immunoblots shown are representative images from ≥2 independent experiments.