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. 2018 Mar 26;115(15):E3436–E3445. doi: 10.1073/pnas.1717423115

Fig. 5.

Fig. 5.

USP7 binding and cell cycle progression is facilitated by NRTK-dependent tyrosine phosphorylation of SPRR2B. (A) Fractionation of Flag-SPRR2B–transfected NIH 3T3 cells and immunoprecipitation of Flag followed by immunoblot for phosphotyrosine. Representative of three independent experiments. Loading controls are HDAC1 (nucleus) and SOD1 (cytoplasm). (B) NIH 3T3 cells transfected with HA-USP7 and Flag-SPRR2B were treated with saracatinib (50 nM) with or without TGF-β1/H2O2. Coimmunoprecipitation of HA reveals stimulation of Flag-SPRR2B interaction by TGF-β1/H2O2 is blocked by saracatinib treatment. TCL, total cell lysate. (C) Quantification of B. n = 3. (D) Coimmunoprecipitation of HA-USP7/Flag-SPRR2B (wild type or Y67F) reveals USP7 association and MDM2 accumulation is dependent upon SPRR2B-Y67 phosphorylation. (E) Quantification of D. n = 3. (F) Expression of genes encoding cell cycle regulators was evaluated by qRT-PCR in CFs transfected with SPRR2B or SPRR2B-Y67F with or without TGF-β1/H2O2. n = 6. qRT-PCR data normalized to Gapdh and analyzed by one-way ANOVA. (G) CyQuant assay was used to evaluate proliferation of CFs in response to SPRR2B or SPRR2B-Y67F transfection with or without TGF-β1/H2O2. n = 8. Analyzed by two-way ANOVA with Bonferroni post hoc. All data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #P < 0.05 compared with second condition.