Fig. 4

ISG15 directly binds to SPD and SPM. (A) Purification of mouse ISG15 (mISG15) and human ISG15 (hISG15). GST-tagged mISG15 and hISG15 are expressed in Escherichia coli and purified using Glutathione sepharose 4B beads. GST tag is removed by PreScission Protease, and purity is confirmed by SDS-PAGE and Coomassie brilliant blue (CBB) staining. (B) mISG15 directly binds to SPD and SPM. Recombinant mISG15 is pulled down using SPD- or SPM-conjugated sepharose, followed by immunoblotting with an anti-mISG15 antibody. Representative data from five independent experiments. (C) hISG15 directly binds to SPD and SPM. Recombinant hISG15 is pulled down using SPD- or SPM-conjugated sepharose, followed by immunoblotting using an anti-hISG15 antibody. Representative data of three independent experiments. (D) Amino acid sequence alignment of hISG15 and mISG15. The numbers indicate amino acid positions in each protein. Acidic amino acids are represented in bold font. Loss of acidic amino acids in hISG15 is boxed, and conserved acidic amino acid sequences are underlined in red. Amino acids that are removed when both ISG15 proteins mature are underlined in black. Both ISG15 ends terminate with -LRLRGG. (E) Purification of wild-type(WT) and mutant mISG15. GST-tagged mISG15 are expressed in E. coli and purified using Glutathione sepharose 4B beads. GST tag is removed by PreScission Protease, and purity is confirmed by SDS-PAGE and CBB staining. (F) D103A or E133L mutation of mISG15 reduced interaction with SPD and SPM. Recombinant mISG15(WT or mutant) is pulled down using SPD- or SPM-conjugated sepharose, followed by immunoblotting using anti-mISG15 antibody. Representative data from five independent experiments. (G) Quantification of SPD- or SPM-bound mISG15 in (F). The signal intensity of SPD- or SPM-bound mISG15(WT) is set as 1. Data are expressed as the mean ± standard deviation of five independent experiments. The means of different groups are compared using the Student’s t-test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively. (H) Purification of mutant hISG15. GST-tagged hISG15 are expressed in E. coli and purified using Glutathione sepharose 4B beads. The GST tag is removed by PreScission Protease, and purity is confirmed by SDS-PAGE and CBB staining. (I) D119/120A mutation of hISG15 reduces interaction with SPD, and E132A/D133A mutation increases interaction with SPM. Recombinant hISG15 (WT or mutant) is pulled down using SPD- or SPM-conjugated sepharose, followed by immunoblotting with an anti-hISG15 antibody. Representative data from five independent experiments. (J) Quantification of SPD- or SPM-bound hISG15 from (I). The signal intensity of control pulldown is subtracted from that of SPD- or SPM-bound hISG15. The signal intensity of SPD- or SPM-bound hISG15(WT) is set to 1. Data are expressed as the mean ± standard deviation of five independent experiments. The means of different groups are compared using the Student’s t-test. p values greater than 0.05 are labeled as N.S., whereas p values less than 0.05 and 0.01 are labeled as * and **, respectively.