Fig. 3: TRIM22 directly binds to PHLPP2 and induces its ubiquitin-mediated degradation.
From: TRIM22 induces cellular senescence by targeting PHLPP2 in hepatocellular carcinoma
A Analysis of PHLPP2 protein stability by Western blotting. TRIM22-overexpressed HepG2 cells were treated with cycloheximide (CHX) for the indicated times, harvested, and analyzed by Western blotting. Data are presented as mean ± SD (unpaired two-tailed t-test, **P = 0.0047, t = 5.708; *P = 0.0208, t = 3.700; **P = 0.0021, t = 7.040, n = 3). B Western blotting analysis of TRIM22-overexpressed HepG2 cells treated with the proteasomal degradation inhibitor, MG132, or the lysosomal inhibitor, CQ. C Co-immunoprecipitation (Co-IP) assays for the interaction between TRIM22 and PHLPP2. HepG2 cells were transfected with EV or TRIM22-Myc and then treated with 20 μM MG132 for 4 hrs. The cells were subjected to IP using anti-Myc (left) or anti-PHLPP2 (right) antibodies. Immunoprecipitates were analyzed by Western blotting using the indicated antibodies. D Proximity ligation assay (PLA) for cytoplasmic interaction between TRIM22 and PHLPP2 using each antibody. TRIM22-Myc-overexpressing HepG2 cells were treated with 20 μM MG132 for 4 hrs and subjected to PLA. The red spots indicate TRIM22-PHLPP2 interactions. Nuclei were stained with DAPI. Scale bars, 20 μm. E Schematic showing the domain structures of TRIM22 Wt and Muts (ΔRING, ΔBBox, ΔCC, or ΔSPRY) (Upper). HepG2 cells were transfected with TRIM22 Wt or Muts (ΔRING, ΔBBox, ΔCC, or ΔSPRY). IP was performed and immunoprecipitates were subjected to Western blot analysis. F Schematic showing the domain structures of PHLPP2 Wt and PHLPP2 ΔC-terminal (ΔCTD) (Upper). Flag-tagged PHLPP2 Wt or PHLPP2 ΔCTD was co-transfected with TRIM22 Wt into HepG2 cells. PHLPP2 Wt or PHLPP2 ΔCTD was immunoprecipitated using Flag antibody, and the immunoprecipitates were analyzed using Western blotting (Bottom). G Ubiquitination assays of PHLPP2 in TRIM22 Wt or Muts (ΔRING, ΔBBox, ΔCC, or ΔSPRY)-overexpressed HepG2 cells. H Co-IP and Western blot analysis of PHLPP2 Wt or PHLPP2 ΔCTD K48 ubiquitination in TRIM22 Wt-overexpressed HepG2 cells.
