Journal: PLoS ONE

Article Title: Covalent Protein Modification with ISG15 via a Conserved Cysteine in the Hinge Region

doi: 10.1371/journal.pone.0038294

Figure Lengend Snippet: (A) HeLa cells were transiently transfected with pCMV2a-Flag-UBE1L, pCMVb-HA-UbcH8 and pCMVb-MRGS-His-ISG15 WT or different mutants. 24 h post-transfection, the cells were collected and lysed with or without 2-ME. (B) Isolation of ISG15 conjugates by metal-chelate pull-down. HeLa cells were transiently transfected with pCMV2a-Flag-UBE1L and pCMVb-MRGS-His-ISG15 WT and different mutants. 24 h post-transfection the cells were collected and lysed without 2-ME. The metal-chelate pull-downs and the SDS-PAGE were carried out under denaturing conditions without 2-ME. The asterisk indicates an unspecific band. (C) Analysis of ISG15 modification of UBE1L by anti-FLAG pull-down. HeLa cells were transiently transfected with pCMV2a-Flag-UBE1L, pCMVb-HA-UbcH8 and pCMVb-MRGS-His-ISG15 WT and different mutants. 24 h post-transfection, the cells were collected and lysed. Flag-UBE1L was immunoprecipitated with anti-FLAG M2 Sepharose as described. SDS-PAGE was carried out in presence of 2-ME. Equal protein loading was verified by immunoblotting against alpha-tubulin.

Article Snippet: The cDNAs encoding for ISG15 (IMAGE ID IRAUp969E1149D, GenBank identification number (GI): BC009507), UbcH8 (UBE2H) (IMAGE ID IRAUp969G0731D, GI: BC006277), UBE1L (UBA7) (IMAGE ID IRAUp969H0433D, GI: BC006378), Ubc13 (IMAGE ID IRATp970B041D, GI: BC003365) and MxA (IMAGE ID IRATp970B1055D6, GI: BC032602) were purchased from ImaGenes (now Source BioScience).

Techniques: Transfection, Isolation, SDS Page, Modification, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Covalent Protein Modification with ISG15 via a Conserved Cysteine in the Hinge Region

doi: 10.1371/journal.pone.0038294

Figure Lengend Snippet: HeLa cells were transiently transfected with pCMVb-HA-Ubc13 WT, pCMVb-HA-Ubc13 C87G mutant or pCMVb-HA-Ubc13 K92R mutant and other vectors as shown in the figure. 24 h post-transfection the cells were collected and lysed. Anti-HA immunoprecipitations were performed as described. Immunoblotting against the S-tag shows the expression levels of both UBE1L and UbcH8. SDS-PAGE was carried out in presence of 2-ME. The asterisk indicates monoubiquitylated Ubc13 and the hash dimers of Ubc13.

Article Snippet: The cDNAs encoding for ISG15 (IMAGE ID IRAUp969E1149D, GenBank identification number (GI): BC009507), UbcH8 (UBE2H) (IMAGE ID IRAUp969G0731D, GI: BC006277), UBE1L (UBA7) (IMAGE ID IRAUp969H0433D, GI: BC006378), Ubc13 (IMAGE ID IRATp970B041D, GI: BC003365) and MxA (IMAGE ID IRATp970B1055D6, GI: BC032602) were purchased from ImaGenes (now Source BioScience).

Techniques: Transfection, Mutagenesis, Western Blot, Expressing, SDS Page

Journal: PLoS ONE

Article Title: Covalent Protein Modification with ISG15 via a Conserved Cysteine in the Hinge Region

doi: 10.1371/journal.pone.0038294

Figure Lengend Snippet: HeLa cells were transiently transfected with ISG15 or control miRNA vectors in which the miRNA is coded from the 3′-UTR of GFP. 12 h post-transfection the cells were induced with IFN-β for another 12 h. (A) After a total of 24 h post-transfection, cells were fixed and subjected to immunofluorescent staining with rabbit polyclonal ISG15 antibody and secondary Alexa Fluor 647 donkey-anti-rabbit antibody (red) and compared to the GFP signal (green). (B) GFP-positive cells were FACS sorted, lysed in urea buffer and analysed for ISG15 downregulation by Western blotting with reducing agent. (C) HeLa cells from the experiment in (B) were additionally co-transfected with pCMV2a-Flag-UBE1L, pCMV2b-Flag-MxA, pCMVb-HA-Ubc13 WT or C87G mutant or and analysed for substrate specific ISGylation by Western blotting in presence of reducing agent. Equal loading of total protein was verified by anti-alpha-tubulin immunoblotting. (D) FACS sorted GFP-positive HeLa cells from the experiment in (B) were lysed with and without 2-ME and analysed by Western blotting against endogenous MxA.

Article Snippet: The cDNAs encoding for ISG15 (IMAGE ID IRAUp969E1149D, GenBank identification number (GI): BC009507), UbcH8 (UBE2H) (IMAGE ID IRAUp969G0731D, GI: BC006277), UBE1L (UBA7) (IMAGE ID IRAUp969H0433D, GI: BC006378), Ubc13 (IMAGE ID IRATp970B041D, GI: BC003365) and MxA (IMAGE ID IRATp970B1055D6, GI: BC032602) were purchased from ImaGenes (now Source BioScience).

Techniques: Transfection, Control, Staining, Western Blot, Mutagenesis