Fig 2: MKRN2 is a RING finger-containing protein that interacts with PDLIM2.(a) Schematic diagram of the structure of mouse MKRN2. Black boxes: C3H-type zinc finger domain (C3H), striped box: unique Cys-His motif (Cys-His), gray box: RING finger domain (RING). (b) Western blot analysis of MKRN2 expression in primary immune cells and MEFs. Whole cell lysates were subjected to immunoblot (IB) with anti-MKRN2 and HSP90 antibodies. MEF: mouse embryonic fibroblasts. Western blots are representative of at least three independent experiments. (c) Subcellular localization of MKRN2 protein. Splenic CD11c+ cells, MEF, NIH3T3, HeLa and RAW264.7 cells were fractionated into cytoplasmic (C) and nuclear (N) fractions, which were then analyzed by Western blotting with the MKRN2 antibody. The purity of the fractionations was confirmed by cdc37 (cytoplasm) or LSD1 (nucleus) antibody reactivity. Western blots are representative of at least three independent experiments. (d) MKRN2 interacts with PDLIM2 through LIM domain of PDLIM2. 293 T cells were transfected with His-tagged PDLIM2 or the PDLIM2 mutant lacking the LIM domain (?LIM) along with or without c-Myc-tagged MKRN2. Whole cell extracts were immunoprecipitated with a c-Myc antibody, and immunoblotted with anti-His. Western blots are representative of at least three independent experiments.

Fig 3: PDLIM7 is a ubiquitin E3 ligase that inhibits NF-κB activation. (A,B) Ubiquitination assay for p65 in 293T cells transfected with plasmids encoding histidine-tagged ubiquitin (His-Ub), p65 and PDLIM1-7 or empty vector (–). Ubiquitinated proteins were purified with Ni beads. Polyubiquitination of p65 (A) or autoubiquitination of PDLIM1-7 (B) were analyzed by immunoblot (IB) with anti-p65 or anti-c-Myc antibody, respectively. Western blots are representative of two independent experiments. (C) Western blot analysis of PDLIM7 expression in primary immune cells, including splenic CD4+T, CD8+T, CD19+B cells, Flt3L-differentiated bone marrow derived dendritic cells (BMDC) and peritoneal macrophages. Whole cell lysates were subjected to immunoblot with anti-PDLIM7 and HSP90 antibodies. Western blots are representative of four independent experiments. (D) Flt3L-defferentiated bone-marrow derived dendritic cells (BMDC) were stimulated without or with LPS (100 ng/ml) for 5 h and fractionated into cytoplasmic (C) and nuclear (N) fractions, which were then analyzed by Western blotting with anti-PDLIM7 antibody. The purity of the fractionations was confirmed by blotting with anti-cdc37 (cytoplasm) or anti-LSD1 (nucleus) antibody. Western blots are representative of three independent experiments. (E) Luciferase activity in MEFs transfected with an ELAM−1 luciferase reporter construct (ELAM-1-luc) and pRL-Null renilla constructs along with or without a plasmid encoding PDLIM7, then left untreated or treated with LPS (2 or 10 ng/ml) for 5 h. Data are representative of four independent experiments. (F) Luciferase activity in MEFs transfected with ELAM-1-luc and pRL-Null with or without plasmids encoding NF-κB p65 in the absence or presence of increasing amounts (wedge) of PDLIM7. Data are representative of four independent experiments. Data in (E,F) are means ± SD. **P < 0.01.

Fig 4: Enhanced p65-mediated inflammatory responses due to MKRN2 deficiency.(a) Reduction of MKRN2 protein amount in bone marrow-derived dendritic cells, transfected with siRNA specific for MKRN2, analyzed by immunoblot with anti-MKRN2. Data are representative of three independent experiments. (b) Western blot analysis for cytoplasmic and soluble and insoluble nuclear p65 in bone marrow-derived dendritic cells transfected with control or MKRN2-specific siRNA, then left unstimulated or stimulated with the indicated concentration of LPS for 1 h and analyzed by antibodies against the indicated targets. The purity of the fractionations was confirmed by anti-cdc37 (cytoplasm), anti-LSD1 (nuclear soluble) or anti-Lamine B (nuclear insoluble) antibody staining. Western blots are representative of three independent experiments. (c) Real-time RT-PCR analysis of LPS-inducible gene expression in bone marrow-derived dendritic cells transfected with control or MKRN2-specific siRNA, then left unstimulated or stimulated with the indicated concentration of LPS for 5 h. (d) Real-time RT-PCR analysis of p65 gene expression in bone marrow-derived dendritic cells transfected with control or MKRN2-specific siRNA, then left unstimulated or stimulated with the indicated concentration of LPS for 5 h. (e) Western blot analysis for cytoplasmic and soluble and insoluble nuclear p65 in NIH3T3 cells transfected with control or MKRN2-specific siRNA, then left unstimulated or stimulated with LPS (10 ng/ml) for 1 h and analyzed by antibodies against the indicated targets. The purity of the fractionations was confirmed by anti-cdc37 (cytoplasm), anti-LSD1 (nuclear soluble) or anti-Histone H3 (nuclear insoluble) antibody staining. Western blots are representative of three independent experiments.