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Figure 1 | Molecular Cancer

Figure 1

From: ISG20L1 is a p53 family target gene that modulates genotoxic stress-induced autophagy

Figure 1

ISG20L1 antibody production and protein analysis. (a) Western analyses demonstrated the polyclonal antibody generated was able to detect endogenous ISG20L1. H460 cells were reverse transfected with Dharmacon siRNA control (Con) or Dharmacon ISG20L1 Ontarget Plus pool and grown for 72 h, at which point the cultures were treated with 8 Gy ionizing radiation and harvested 24 h after treatment (left panel). Ectopic expression of a FLAG-ISG20L1 detected by our ISG20L1 antibody serves as further positive control. Asterisks mark nonspecific bands and the arrow indicates ISG20L1. Western is representative of three independent experiments. (b) ISG20L1 is localized to the nucleolus and perinucleolar region. RKO cells were transfected with either empty vector (Vector Control) or FLAG-ISG20L1 and immunofluorescence analysis was performed 24 h later using a FLAG antibody or the ISG20L1 antibody and the nuclei co-stained with DAPI. Views of individual and merged stainings are shown. Insets are further magnification of individual cells to highlight the perinucleolar staining of ISG20L1. Immunofluorescence was performed on four independent transfections using ISG20L1 and FLAG antibodies. (c) p53-dependent regulation of ISG20L1 is shown by Western analysis. NHEK cells were infected with shRNA lentiviral constructs expressing either shRNA to p53 or a scrambled shRNA (Con) and then treated with cisplatin (5 μg/mL) for 6 h. Protein lysates were prepared and analyzed for p53, ISG20L1, and actin. The Western blot is representative of three independent experiments.

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