Pa5 20390

Sample collection and western blotting were performed in accordance with previously described methods [29 (link)]. Rabbit monoclonal anti-RIG-I antibody (#3743S; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-IFN-α 11 antibody (bs-7023R; Bioss Antibodies, Woburn, MA, USA), rabbit polyclonal anti-IFN-β antibody (GTX37658; GeneTex, Irvine, CA, USA), Rabbit monoclonal anti-RIG-I antibody (#700366; Invitrogen, Carlsbad, CA, USA), rabbit polyclonal anti-IFN-α2 antibody (ab193055; Abcam, Cambridge, UK), rabbit polyclonal anti-IFN-β antibody (PA5-20390; Invitrogen), rat monoclonal anti-human klotho antibody (KO603; TransGenic, Fukuoka, Japan), mouse monoclonal anti-β-actin antibody (A5316; Sigma-Aldrich, St. Louis, MO, USA), and mouse monoclonal anti-α-tubulin antibody (T9026; Sigma-Aldrich) were used as primary antibodies. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Dako, Glostrup, Denmark) and goat anti-mouse immunoglobulin G (Dako) were used as secondary antibodies. SuperSignal West Dura and the Pico system (Thermo Fisher Scientific) were used to detect signals. The intensity of each band was measured by ImageJ software (version 1.47v; National Institutes of Health, Bethesda, MD, USA) and normalized to the level of either β-actin or α-tubulin.

Immunohistochemical staining was performed in accordance with previously described methods [27 (link)]. As primary antibodies, the following products were applied: rabbit monoclonal anti-RIG-I antibody (#700366; Invitrogen), rabbit polyclonal anti-IFN-α 2 antibody (ab193055; Abcam), rabbit polyclonal anti-IFN-β antibody (PA5-20390; Invitrogen), and rat monoclonal anti-human klotho antibody (KO603; TransGenic). RIG-I-, IFN-α/β-, and klotho-positive areas were quantified as the average of 20 randomly selected fields with ImageJ software.

Immunostaining was carried out in accordance with previously described methods [27 (link)]. The following primary antibodies were used: rabbit monoclonal anti-RIG-I antibody (#700366; Invitrogen), mouse monoclonal anti-IFN-α antibody (sc-373757; Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit polyclonal anti-IFN-β antibody (PA5-20390; Invitrogen). RIG-I- and IFN-α/β-positive areas were quantified as the average of five randomly selected fields with ImageJ software.