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. Author manuscript; available in PMC: 2021 Nov 16.
Published in final edited form as: Oncogene. 2021 Sep 23;40(44):6235–6247. doi: 10.1038/s41388-021-02017-8

Fig. 5. GDI2 is ISGylated and can regulate pAKT levels.

Fig. 5

(A) Label-free quantification (LFQ) values for GDI2 obtained from ISG15 pull-downs of the indicated clones, protein identification and quantification were performed using MaxQuant. (B) Analysis of GDI2 ISGylation using crUBC8 and WT cells transfected with empty vector, Step-tactin-ISG15, MYC-DDK-GDI2 or both, and subjected to a Step-tactin pulldown. WB show the pulldown and a 5% of the total lysates (Input). Arrow indicates ISGylated GDI2. (C) WB of WT and crISG15 cells transfected with MYC-DDK-GDI2, after 48h cells were lysed and subjected to an anti-Flag pull-down. Blots show the pulldowns and a 5% of the total lysates (Input). (D) Analysis of putative GDI2 ISGylation sites. WT cells were transfected with Strep-tactin ISG15, MYC-DDK-GDI2, or Strep-tactin ISG15 with either MYC-DDK-GDI2wt or the indicated GDI2 mutants. After 48h, cells were lysed and subjected to a Strep-tactin pull-down. The ISGylation status of the different GDI2 mutants was measured by determining the levels of GDI2 co-precipitated with ISG15. Blots show the results of the pulldown and a 5% of the total lysates (Input). (E) Analysis of Akt activation in crGDI2 by WB of WT and crGDI2 cells stimulated with EGF 10 ng/ml for 10 min. pAkt is pSer473