Rabbit anti stat1α

Rabbit anti-Stat1α (Santa Cruz Biotechnology, TX, USA) was used for the immunoprecipitation and rabbit anti-IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) was used as a control antibody. Chromatin immunoprecipitation (Ch-IP) was carried out as described [12 (link)], with the exception that the DNA was purified using the PCR Purification Kit (Promega, Fitchburg, WI, USA) and subjected to PCR with the specific primers shown in Supplementary Table 1. A primers set that amplifies the STAT-1 binding sequence on the interferon gamma (IFNG) promoter was used as positive control. A primers set that amplifies a non-specific sequence on the CLU promoter was used as negative control.

Immunoblotting was carried out using the following antibodies: mouse anti-Clusterin-α (Santa Cruz Biotechnology, TX, USA, at 1:1,000), rabbit anti-Stat1α (Santa Cruz Biotechnology, TX, USA, at 1:5,000), rabbit anti-p-Stat1 (Santa Cruz Biotechnology, TX, USA, at 1:5,000), rabbit anti-L1 (gift from Dr. Vance Lemmon, University of Miami, FL, USA, at 1:8,000), and mouse anti-α-tubulin (Sigma-Aldrich, Rehovot, Israel, at 1:100,000). Western blots were developed by using the ECL method (Amersham Biosciences, UK). For immunofluorescence, cells cultured on glass coverslips were permeabilized with 0.5% Triton X-100 and fixed with 3% PFA. The same primary antibodies were used for immunofluorescence as for immunoblotting. The secondary antibody used was Cy3-labeled goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). Images were acquired by using Eclipse E1000, Nikon objectives 60x/1.4 NA with a camera (ORCA-ER; Hamamatsu) and Volocity acquisition software (Improvision).