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. 2021 Feb 2;12(3):160–172. doi: 10.18632/oncotarget.27869

Figure 2. Usp9x deubiquitinates SOX2 and regulates its degradation.

Figure 2

Deubiquitinase or transcription factor knock-down (KD) or overexpression in melanoma cell lines was achieved using lentiviral constructs. (A) After KD of Usp34 with three different shRNA constructs, immunoblot for Usp34 and SOX2 is shown in mutant-BRAF melanoma cell line. (B) KD of Usp9x and immunoblot for the proteins indicated in mutant-BRAF A375melanoma cell lines. (C) Immunoblot for the proteins indicated in NRAS mutant melanoma cell lines with control or Usp9x KD. (D) Exogenously expressed HA-Usp9x (full-length) was co-expressed with FLAG-SOX2 in HEK293T cells. HA (Usp9x) immunoprecipitation was followed by immunoblotting of FLAG-SOX2, total lysate was used as a control. (E) Immunoblot for Usp9x, SOX2, in control and Usp9x KD A375 BRAF-mutant cells treated ± MG132 for 6 h (10 μM). (F) Immunoblot for Usp9x and SOX2 after Usp9x KD in A375 BRAF-mutant cells treated with cycloheximide for 6 hrs. (G) HEK293T cells exogenously expressing FLAG-SOX2 and HA-ubiquitin were subjected to control or Usp9x KD and FLAG immunoprecipitation was followed by HA blotting to detect Ub-SOX2 levels. Immunoblot for FLAG (SOX2) in the pulldowns (top) and input lysate (Usp9x and actin, bottom) is shown. Actin served as a loading control wherever necessary.