Subcellular fractionations were performed per the manufacturer's instructions
(Thermo Scientific, Waltham, MA, USA). Briefly, cells at 80–90% confluence were
lysed in a series of buffers containing protease inhibitors (25X) with CERI (250
μl), CERII (11 μl), or NER (100 μl). Centrifugation steps were performed to
obtain a non-nuclear fraction and an intact nuclear pellet, followed by further
lysing to isolate the nuclear fraction. 30 μg of non-nuclear and nuclear
fractions were utilized for Western blot analysis. Mouse anti-topoisomerase I
(Santa Cruz Biotechnology Santa Cruz, CA) and rabbit anti-GAPDH antibodies (Cell
Signaling Technology, Inc., Danvers, MA) were used to ensure the integrity of
nuclear and cytoplasmic fractions, respectively. Rabbit anti-Calnexin (Santa
Cruz Biotechnology Santa Cruz, CA) was utilized as a control to ensure that the
nuclear fraction was not contaminated with endoplasmic reticulum.
(Thermo Scientific, Waltham, MA, USA). Briefly, cells at 80–90% confluence were
lysed in a series of buffers containing protease inhibitors (25X) with CERI (250
μl), CERII (11 μl), or NER (100 μl). Centrifugation steps were performed to
obtain a non-nuclear fraction and an intact nuclear pellet, followed by further
lysing to isolate the nuclear fraction. 30 μg of non-nuclear and nuclear
fractions were utilized for Western blot analysis. Mouse anti-topoisomerase I
(Santa Cruz Biotechnology Santa Cruz, CA) and rabbit anti-GAPDH antibodies (Cell
Signaling Technology, Inc., Danvers, MA) were used to ensure the integrity of
nuclear and cytoplasmic fractions, respectively. Rabbit anti-Calnexin (Santa
Cruz Biotechnology Santa Cruz, CA) was utilized as a control to ensure that the
nuclear fraction was not contaminated with endoplasmic reticulum.