Journal: The Journal of Experimental Medicine

Article Title: CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts

doi: 10.1084/jem.20150407

Figure Lengend Snippet: CHMP5 down-regulates RANK-mediated NF-κB activation in osteoclasts. (A, B, and H) Fractionation of the CHMP5 protein complex in osteoclasts. RAW264.7 cells were lysed with 1% CHAPS lysis buffer and fractionated by size exclusion chromatography. The fractions were immunoblotted with the indicated antibodies (A). The fraction in the red box was immunoprecipitated with anti-Chmp5 antibody or IgG control antibody along with protein G–conjugated dynabeads, and immunoblotted with the indicated antibodies (B and H). The images in A, B, and H represent two independent experiments. (C) HEK293 cells were transfected with HA-Chmp5 along with vector or Flag tagged-IκBα or -RelA. After 48-h transfection, the cells were lysed, immunoprecipitated with anti-Flag antibody–conjugated agarose, and immunoblotted with the indicated antibodies. (D) RAW264.7 cells were transfected with Chmp5 in a dose-dependent manner along with PBII-luc and Renilla and stimulated with 50 ng/ml of RANKL (top) or RAW264.7 cells expressing control, or Chmp5 shRNA were treated with different concentrations of RANKL (bottom). Relative luciferase activity was normalized to Renilla . *, P < 0.05; ***, P < 0.0001 by a Bonferroni-corrected two-tailed Student’s t test. (E-G) Chmp5 fl/fl and Chmp5 Ctsk BMMs were cultured in the presence of M-CSF and RANKL, and after 3 d total cell lysates were used for an NF-κB DNA-binding assay (E) or immunoblotted with the indicated antibodies (F). Alternatively, BMMs were infected by lentiviruses expressing vector or the super repressor, HA-IκBα-RR, and osteoclast differentiation was measured by TRAP activity (G). **, P < 0.01; ***, P < 0.0001 by a Bonferroni-corrected two-tailed Student’s t test.

Article Snippet: In brief, HEK293 cells or RAW264.7 cells were transfected with a NF-κB–responsive (PBII-luc) or AP1-responsive reporter gene (2xAP1-luc) along with Renilla (Promega).

Techniques: Activation Assay, Fractionation, Lysis, Size-exclusion Chromatography, Immunoprecipitation, Control, Transfection, Plasmid Preparation, Expressing, shRNA, Luciferase, Activity Assay, Two Tailed Test, Cell Culture, DNA Binding Assay, Infection

Journal: The Journal of Experimental Medicine

Article Title: CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts

doi: 10.1084/jem.20150407

Figure Lengend Snippet: CHMP5 and USP15 suppress RANKL-induced NF-κB activation and osteoclast differentiation via stabilization of IκBα. (A) HEK293 cells were transfected with Chmp5 or Usp15 along with Flag-IκBα, His-ubiquitin, and Myc-β-Trcp, treated with 10 µM MG132, and subjected to IκBα ubiquitination (top) and protein expression assays (bottom). (B and C) HEK293 cells were transfected with control vector, Chmp5, or Usp15 along with Flag-IκBα, and IκBα stability was determined by pulse–chase labeling with [ 35 S]-methionine followed by autoradiography and quantified by ImageJ (C). (D and E) RAW264.7 cells expressing control vector, Chmp5, or Usp15 shRNA were stimulated with 25 ng/ml of RANKL at different time points, and immunoblotted with the indicated antibodies (D). IκBα degradation was quantified using ImageJ and normalized to Hsp90 expression (E). (F and G) RAW264.7 cells expressing control vector or Usp15 shRNA were transfected with Chmp5 along with PBII-luc and Renilla (F). Alternatively, RAW264.7 cells were transfected with Usp15 in a dose-dependent manner along with PBII-luc and Renilla (G). Luciferase activity was measured and normalized to Renilla . *, P < 0.05; **, P < 0.01; ***, P < 0.0001 by a Bonferroni-corrected two-tailed Student’s t test. (H) RAW264.7 cells expressing control vector or Usp15 shRNA were cultured in the presence of M-CSF and different concentrations of RANKL. TRAP staining was performed after 6 d. (I) WT BMMs were treated with vehicle or NF-κB inhibitor for 1 h before RANKL stimulation. After 48 h of stimulation with RANKL, Chmp5 and Usp15 expression was analyzed by RT-PCR. ***, P < 0.0001 by a Bonferroni-corrected two-tailed Student’s t test. Data in A–I represent n = 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Bar: (H) 60 µm.

Article Snippet: In brief, HEK293 cells or RAW264.7 cells were transfected with a NF-κB–responsive (PBII-luc) or AP1-responsive reporter gene (2xAP1-luc) along with Renilla (Promega).

Techniques: Activation Assay, Transfection, Ubiquitin Proteomics, Expressing, Control, Plasmid Preparation, Pulse Chase, Labeling, Autoradiography, shRNA, Luciferase, Activity Assay, Two Tailed Test, Cell Culture, Staining, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts

doi: 10.1084/jem.20150407

Figure Lengend Snippet: CHMP5 mediates deubiquitination of the VCP/p97 complex via USP15. (A) GST or GST-Chmp5 was incubated with the purified proteins, immunoprecipitated with glutathione-agarose, and immunoblotted with the indicated antibodies (top). Alternatively, GST or GST-VCP was incubated with purified CHMP5 protein (bottom). Input indicates loading controls for USP15, VCP, and CHMP5. (B) RAW264.7 cells expressing a control vector or Chmp5 shRNA were cultured with 5 µg of RANKL for 2 d before treatment with 10 µM MG132. Cell lysates were immunoprecipitated with anti-Vcp antibody and protein G–conjugated dynabeads and immunoblotted with the indicated antibodies. (C) HEK293 cells were transfected with Myc-VCP and HA-ubiquitin in the absence or presence of CHMP5 or USP15. 24 h later, cells were treated with 10 µM MG132 and subjected to ubiquitination of Myc-VCP. (D and E) HEK293 cells were transfected with HA-CHMP5 (D) or USP15 (E) and His-ubiquitin along with vector, Myc-VCP (WT), or Myc-VCP mutants (R155H, A232E). 24 h later, cells were treated with 10 µM MG132, immunoprecipitated with anti-Myc antibody–conjugated agarose, and immunoblotted with the indicated antibodies. (F) HEK293 cells were transfected with Myc-VCP (WT) or Myc-VCP mutants (R155H, A232E) and HA-ubiquitin in the absence or presence of CHMP5 or USP15. 24 h later, cells were treated with 10 µM MG132 and subjected to ubiquitination of Myc-VCP. (G) HEK293 cells were transfected with vector, Myc-VCP (WT), or Myc-VCP mutants (R155H, A232E) along with Flag-RANK, PBII-luc and Renilla in the absence or presence of USP15, and 24 h later, luciferase activity was measured and normalized to Renilla . **, P < 0.01 by a Bonferroni-corrected two-tailed Student’s t test. Data in A–G represent n = 2 independent experiments. **, P < 0.01.

Article Snippet: In brief, HEK293 cells or RAW264.7 cells were transfected with a NF-κB–responsive (PBII-luc) or AP1-responsive reporter gene (2xAP1-luc) along with Renilla (Promega).

Techniques: Incubation, Purification, Immunoprecipitation, Expressing, Control, Plasmid Preparation, shRNA, Cell Culture, Transfection, Ubiquitin Proteomics, Luciferase, Activity Assay, Two Tailed Test