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. 2016 Aug 22;7:12513. doi: 10.1038/ncomms12513

Figure 1. ATM/ATR kinases induce the expression of ISG15-conjugating system.

Figure 1

p53+/+ HCT116 cells were treated with 1 μM doxorubicin (DOX) (a), 0.25 μM camptothecin (CPT) (b) and 30 J m−2 ultraviolet (UV) (c). Immediately after the treatment, the cells were incubated with 5 mM caffeine for increasing periods. The cells were washed with iced PBS, and lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM NEM, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF, 0.2% (v/v) Triton X-100 and 1 X protease inhibitor cocktail. The samples were centrifuged for 30 min at 16,000g and the supernatant fractions (cell lysates) were subjected to immunoblot with anti-ISG15, anti-UBE1L anti-UBCH8, anti-EFP, anti-phospho-p53 (p-p53), anti-p53, anti-phospho-Chk1 (p-Chk1) and anti-β-actin antibodies (from the top to the bottom, respectively).