Journal: Frontiers in Immunology

Article Title: PDLIM7 Synergizes With PDLIM2 and p62/Sqstm1 to Inhibit Inflammatory Signaling by Promoting Degradation of the p65 Subunit of NF-κB

doi: 10.3389/fimmu.2020.01559

Figure Lengend Snippet: PDLIM7 promotes p65 degradation in a LIM3 domain-dependent manner. (A) PDLIM7 interacts with p65 but not with p50 subunit of NF- κ B. 293T cells were transfected with a FLAG-tagged p65 or p50 expression plasmid along with or without PDLIM7. Whole cell extracts were immunoprecipitated with anti-c-Myc and immunoblotted with anti-FLAG. Western blots are representative of four independent experiments. (B) Effect of PDLIM7 on cytoplasmic, soluble and insoluble nuclear p65 in NIH3T3 cells transfected with plasmids encoding FLAG-tagged p65 and increasing amounts of c-Myc-tagged PDLIM7. Cells were subjected to fractionation and analyzed with the indicated antibody. Western blots are representative of three independent experiments. (C) Western blot analysis for soluble and insoluble nuclear p65 in NIH3T3 cells transfected with plasmids encoding p65 without or with PDLIM7, then left untreated or treated for 4 h with MG132 (10 μM) and analyzed as in (B) . (D) NIH3T3 cells were transfected with FLAG-p65 along with or without c-Myc-PDLIM7, left untreated or treated for 4 h with MG132 (10 μM) or bafilomycin (100 μM). Cells were subjected to fractionation and analyzed with anti-FLAG antibody. Western blots are representative of three independent experiments. (E) Ubiquitination assay for p65 in 293T cells cotransfected with plasmids encoding His-tagged ubiquitin (His-Ub) and p65, together without or with wild-type or ΔLIM3 PDLIM7 and analyzed as in . Western blots are representative of three independent experiments. (F) Effect of ΔLIM3 PDLIM7 on cytoplasmic, soluble and insoluble nuclear p65 in NIH3T3 cells transfected with plasmids encoding p65, together without or with wild-type or ΔLIM3 PDLIM7 and analyzed as in (B) . Western blots are representative of five independent experiments. (G) Luciferase activity in MEFs transfected with an ELAM-1-luc and pGL4.32-Null renilla construct with or without plasmids encoding p65 in the absence or presence of expression plasmids encoding wild-type or ΔLIM3 PDLIM7. Data are representative of three independent experiments. Shown are the mean values ± SD. ** P < 0.01.

Article Snippet: FLAG-tagged p50 expression plasmid (p50 cFLAG pcDNA3) was purchased from Addgene (#20018).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Fractionation, Ubiquitin Assay, Luciferase, Activity Assay, Construct