Journal: Cell Death & Disease

Article Title: Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon

doi: 10.1038/s41419-017-0138-9

Figure Lengend Snippet: a – e All samples were transfected with plasmids encoding UBE1L and Myc-tagged UbcH8. a HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP and/or FLAG-HERC5. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by immunoblotting with anti-Xpress or anti-HERC5 antibody. b Cells were transfected for 36 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, or Myc-EFP, alone or in combination. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-Myc or anti-Xpress antibody. c HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, FLAG-HERC5-WT, or its catalytically inactive mutant FLAG-HERC5-CA, alone or in combination. Cell lysates were subjected to NTA pull-down (PD: NTA) under denaturing conditions followed by western blotting with anti-FLAG or anti-Xpress antibody. d Cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, Myc-EFP-WT, or Myc-tagged catalytically inactive EFP mutant Myc-EFP-CS, alone or in combination. Cell lysates were subjected to NTA pull-down, as in c . e HEK293 cells were transfected for 48 h with nonspecific control scrambled siRNA (NC), HERC5 -siRNA (H5), or plasmid encoding Xpress-His-CHIP or FLAG-ISG15, alone or in combination. Cell lysates were subjected to NTA pull-down as in b . f Cells were transfected for 36 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, or Flag-HHARI, alone or in combination. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-Flag or anti-Xpress antibody. Asterisk indicates IgG heavy chains, and closed arrowhead indicates predicted or right size band

Article Snippet: Polyclonal anti-HERC5 antibodies were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis, Control

Journal: Cell Death & Disease

Article Title: Covalent ISG15 conjugation to CHIP promotes its ubiquitin E3 ligase activity and inhibits lung cancer cell growth in response to type I interferon

doi: 10.1038/s41419-017-0138-9

Figure Lengend Snippet: a , b All samples were transfected with plasmids encoding UBE1L and Myc-UbcH8. a HEK293 cells were transfected for 24 h with plasmid encoding HA-CHIP, FLAG-ISG15-GG, or FLAG-ISG15-AA, alone or in combination. Cells were treated for an additional 6 h with 10 μM MG132. Cell lysates were immunoprecipitated with anti-HA antibody, followed by western blotting with anti-ubiquitin antibody. Tubulin served as a loading control. b HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP, FLAG-ISG15, FLAG-HERC5-WT, or FLAG-HERC5-CA, alone or in combination, and treated for an additional 6 h with 10 μM MG132. Cell lysates were immunoprecipitated with anti-Xpress antibody, followed by western blotting with anti-ubiquitin antibody. Actin served a as loading control. c A549 cells were treated for 48 h with vehicle (−) or IFN-α (1000 U/ml). Immunoprecipitation of cell lysates was performed with preimmune IgG or anti-CHIP antibody, followed by western blotting with anti-ubiquitin or anti-CHIP antibody. Tubulin served as a loading control

Article Snippet: Polyclonal anti-HERC5 antibodies were purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Ubiquitin Proteomics, Control