Figure 2:

USP18 upregulated ATGL protein by removing the ISG15 conjugate from this substrate. (A) Knock-down of USP18 by individual siRNAs was independently achieved in ED1 and A549 cancer cell lines and this led to decreased ATGL expression, with expression profiles quantified relative to actin expression and normalized to control (inactive) siRNAs independently transfected into these respective lung cancer cell lines. Real-time qPCR assays were done to validate siRNA knockdown of USP18. (B) Gain of USP18 expression was achieved independently in murine ED1 lung cancer and KHC2 leiyomyosarcoma cell lines and this led to increased ATGL protein expression. (C) Gain of expression of the E1-like ubiquitin activating ligase UBE1L conferred reduced ATGL protein expression. Quantification was relative to actin expression as normalized to the empty vector transfected into these respective lung cancer cell lines. (D) Immunoprecipitation assays confirmed that ISG15 and ATGL form conjugated protein, as shown in this panel. The abbreviation NS refers to non-specific and IB indicates immunoblot while IP is immunoprecipitation. The quantification of immunoblots shows the average of three independent experiments. The symbols refer to * P < 0.05 and ** P < 0.01, respectively. Representative immunoblots are displayed.