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. 2018 Jan 24;9(2):97. doi: 10.1038/s41419-017-0138-9

Fig. 4. ISGylation increases CHIP autoubiquitination.

Fig. 4

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