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

Fig. 4.

Fig. 4.

SPRR2B is a binding partner for the MDM2 deubiquitinase USP7 in CFs. (A) qRT-PCR for Sprr2b and genes encoding keratinocyte proteins in primary CFs and DFs with or without TGF-β1/H2O2. (B) Schematic of human SPRR2B protein, highlighting predicted protein–protein interactions related to cell cycle control. The predicted USP7 interaction domain contains a canonical tyrosine phosphorylation site at Y67. (C) Fluorescent detection of phalloidin (green) SPRR2B (red) and nuclei (blue) by confocal microscopy reveals both cytoplasmic and nuclear localization of SPRR2B in CFs. (Scale bar: 10 μm.) (D) NIH 3T3 cells were cotransfected with HA-tagged USP7 with or without Flag-tagged SPRR2B. Immunoprecipitation of HA-USP7, followed by immunoblotting for Flag-SPRR2B or endogenous MDM2 revealed complex formation particularly in the presence of TGF-β1/H2O2. (E) Quantification of D, analyzed by two-way ANOVA with Bonferroni post hoc (n = 3). qRT-PCR data normalized to Gapdh. All data represent mean ± SEM. *P < 0.05, ***P < 0.001 compared with control. #P < 0.05 compared with second condition.