Fig. 3. Identification of covalent ISG15 conjugation sites in CHIP.

a Schematic diagram of all lysine residues in full-length CHIP. b–e HEK293 cells were transfected for 24 h with plasmid encoding Xpress-His-CHIP (WT), the indicated point mutants, or FLAG-ISG15, alone or in combination. Cell lysates were subjected to NTA pull-down (PD: NTA), followed by western blotting with anti-FLAG or anti-Xpress antibody. In the CHIP-3KR mutant, the three indicated lysine residues have been substituted with arginines (K143/144/145 R). The CHIP-4KR mutant has the same point mutations, plus substitution of K287 with arginine (K143/144/145/287 R). All samples in b–e were transfected with plasmids encoding UBE1L and Myc-UbcH8. Closed arrowhead indicates predicted or right size band