Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: ( A ) Domain structure of NEMO ( B ) Western blot showing expression of pMX-NEMO WT (NM) and pMX-NEMO mutants (NM-K270A, NEMO-D304N and NM-K319A). ( C ) BMMs from WT and ( LysM-cre-NEMO f/f ) NEMO-cKO mice were transduced with viral particles (generated by transfecting pMX- retroviral vectors in PLAT-E cells) expressing NEMO WT (NM-WT) and NEMO K270A (NM-KA) and cultured in the presence of MCSF (10 ng/ml) and RANKL (50 ng/ml). ( D ) Representative TRAP staining for osteoclast (n = 8) and ( D ) quantification of TRAP positive OCs. qPCR analysis for OC marker genes ( E ) TRAP, ( F ) CTSK, ( G ) MMP9, ( H ) β3integrin, ( I ) DC-STAMP and ( J ) NFATC1 (p=0.057). Representative data (n = 3 independent experiments). ( K ) BMMs from RelA_luc reporter mice expressing NM-WT and NM-KA were cultured in the presence of MCSF (10 ng/ml) for 3 days followed by RANKL stimulation with RANKL (50 ng/ml) for 6 hr and RelA-luciferase activity measurement (n = 3). pMX-Flag-NEMO WT -RFP (NM-WT), pMX-Flag-NEMO K270A -RFP (NM-KA). (*p<0.05, **p<0.01 and ***p<0.001). Figure 1—source data 1. Western blot showing expression of pMX-NEMOWT (NM) and pMX-NEMO mutants (NM-K270A, NEMO-D304N and NM-K319A). Figure 1—source data 2. qPCR analysis for OC marker genes. Figure 1—source data 3. RelA-luciferase activity.

Article Snippet: Strain, Strain backgroud Mus musculus , RELA (NF-ĸB)-GFP- luciferase reporter , The Jackson Laboratory , NF-ĸB reporter mice , C57BL/6 background.

Techniques: Western Blot, Expressing, Transduction, Generated, Retroviral, Cell Culture, Staining, Marker, Luciferase, Activity Assay

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet: Serum was collected from NM-WT and NM-KA mice (n = 8–10) to measure concentration of inflammatory cytokines ( A ) Interleukin (IL) 1b, ( B ) IL-4, ( C ) IL-6, ( D ) IL-10, ( E ) IL-13, ( F ) IL-17, ( G ) Monocyte chemoattractant protein1 or CCL2, ( H ) Tumor necrosis factor alpha, ( I ) Macrophage colony stimulating factor, ( J ) macrophage Inflammatory protein (MCP)−1 or CCL3, ( K ) keratinocyte chemoattractant or neutrophil activating protein three or CXCL1 and ( L ) granulocyte colony stimulating factor (GCSF). ( M ) BMMs from NM-WT and NM-KA mice were isolated and cultured in the presence of MCSF (10 ng/ml) and RANKL (10 ng/ml). Representative TRAP staining for osteoclast (n = 8) is shown. ( N–R ) Representative qPCR analysis for OC marker genes TRAP, CTSK, β3integrin, DC-STAMP and NFATC1 (n = 3). ( S ) BMMs from NM-WT and NM-KA mice were isolated and cultured in the presence of MCSF (10 ng/ml) four days followed by serum starvation and stimulation with RANKL (50 ng/ml) for different time points (n = 8). Representative western-blot showing activation of p65 (phos-p65/p65 ratio) post RANKL stimulation in BMMs from NM-WT and NM-KA mice. LysM-cre-NEMO-WT-f/f (NM-WT), LysM-cre-NEMO-K270A-f/f (NM-KA) mice. (*p<0.05, **p<0.01 and ***p<0.001). Figure 3—source data 1. Serum concentration of cytokines from NM-WT and NM-KA mice measured by ELISA. Figure 3—source data 2. Representative qPCR analysis for OC marker genes. Figure 3—source data 3. Representative western-blot of p65 (phos-p65/p65 ratio) post RANKL stimulation in BMMs from NM-WT and NM-KA mice.

Article Snippet: Strain, Strain backgroud Mus musculus , RELA (NF-ĸB)-GFP- luciferase reporter , The Jackson Laboratory , NF-ĸB reporter mice , C57BL/6 background.

Techniques: Concentration Assay, Isolation, Cell Culture, Staining, Marker, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Inflammatory osteolysis is regulated by site-specific ISGylation of the scaffold protein NEMO

doi: 10.7554/eLife.56095

Figure Lengend Snippet:

Article Snippet: Strain, Strain backgroud Mus musculus , RELA (NF-ĸB)-GFP- luciferase reporter , The Jackson Laboratory , NF-ĸB reporter mice , C57BL/6 background.

Techniques: Luciferase, Recombinant, Retroviral, Plasmid Preparation, FLAG-tag, Mutagenesis, Construct, Transfection, Activity Assay, BIA-KA, Quantitation Assay, Lysis, Western Blot, Electron Microscopy, Multiplex Assay, Marker, Isolation, SYBR Green Assay, Real-time Polymerase Chain Reaction, Sequencing, Software