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. 2022 Apr 1;132(7):e155224. doi: 10.1172/JCI155224

Figure 6. SENP7-mediated PTEN deSUMOylation facilitates PTEN degradation.

Figure 6

(A) HEK293T cells cotransfected with Flag-tagged PTEN, Ubc9, and RH-SUMO3 in the presence of WT or catalytically inactive (C979S) mutant (M) SENP7 were immunoprecipitated with anti-Flag antibody and assessed by immunoblotting (IB) with anti-SUMO2/3. HA, hemagglutinin; RH, arginine(R)-glycine(G)-serine(S)-histidine. (B) PTEN SUMOylation assays were performed by immunoprecipitating PTEN under denaturing conditions followed by detection of SUMOylated PTEN using anti-SUMO2/3 antibodies in CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. (C) Quantifications of PTEN-SUMO2/3:PTEN levels in CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies (n = 3). (D) Immunoblot analysis of the indicated proteins in CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 1 hour following incubation with CHX (50 μg/mL) for the indicated durations. (E) PTEN ubiquitination assays in CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies in the presence of MG132. (F) Immunoblot analysis of the indicated proteins in CD8+ T cells from WT mice stimulated with anti-CD3 and anti-CD28 antibodies plus LMB for 2 hours. (G) PTEN SUMOylation assays using nuclear and cytoplasmic fractions of WT and KO CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours. (H) Quantification of PTEN-SUMO2/3:PTEN levels in the cytoplasmic fractions of WT and KO CD8+ T cells stimulated with anti-CD3 and anti-CD28 antibodies for 2 hours (n = 4). (I) Immunoblot analysis of the indicated proteins in CD8+ T cells from WT mice stimulated with anti-CD3 and anti-CD28 antibodies plus LMB for 0, 2, and 4 hours. (J and K) PTEN SUMOylation (J) and ubiquitination (K) assays using nuclear and cytoplasmic fractions of CD8+ T cells from WT mice stimulated with anti-CD3 and anti-CD28 antibodies plus LMB for 2 hours. Data are representative of 3 independent experiments and presented as the mean ± SEM. *P < 0.05 and **P < 0.01, by Student’s t test.