Slx4ip

RNA was isolated, quantified, and analyzed as described previously.^{35 (link)} Protein isolation, western blotting analysis and quantification was completed as published previously.^{35 (link)} Primary antibody dilutions are as follows: 1:10,000 GAPDH (Cell Signaling, 2118), 1:3,000 SLX4IP (Sigma, HPA046372), 1:3,000 FLAG (Sigma, F1804), 1:5,000 TRF2 (Novus Biologicals, NB110–57130), 1:1,000 PML (Cell Signaling, E5R8T and Santa Cruz Biotechnology, sc-5621), 1:3,000 p21 (Cell Signaling, 2947).
Coimmunoprecipitation experiments were carried out using the Dynabeads™ Co-Immunoprecipitation Kit according to manufacturer instructions (ThermoFisher Scientific). The provided immunoprecipitation (IP) buffer included was modified with an additional 50 mM NaCl and 0.05% Triton X-100. Cells were grown in 15 cm dishes and scraped using the modified IP buffer, treated with 5 μL of recombinant DNaseI (Sigma), rotated at 4° C for one hour, and cleared by centrifugation. Following protein quantification, 2 mg of protein was incubated with 1.5 mg of Dynabeads coupled with 10 µL of FLAG antibody (Sigma, F1804) or 20 µL of SLX4IP antibody (Sigma, HPA046372) per protocol. After completion, whole cell lysates and coimmunoprecipitation samples were analyzed by western blotting as described above.

Western blotting was completed as described previously (47 (link)). Briefly, 50 μg of lysate was resolved on a 4% to 20% Mini-PROTEAN TGX Gel (Bio-Rad) and transferred to Nitrocellulose Membrane (Millipore) using the Trans-Blot Turbo (Bio-Rad). Blots were blocked in 5% nonfat milk diluted in TBST and incubated overnight at 4°C with primary antibody. Blots were incubated for 1 hour at room temperature with either horseradish peroxidase–conjugated (Santa Cruz Biotechnology), IRDye 800CW (LI-COR), or IRDye 680RD (LI-COR) secondary antibodies diluted to 1:10,000. Development with ProtoGlow ECL Reagent (National Diagnostics) was used when necessary. Blots were imaged on an Odyssey Fc Imaging System (LI-COR), quantified using Image Studio 5.2 software, and normalized to GAPDH. Primary antibody dilutions used are as follows: 1:20,000 GAPDH (Cell Signaling Technology, 2118), 1:5,000 SLX4IP (Sigma, HPA046372), 1:5,000 FLAG (Sigma, F1804), 1:10,000 AR (Santa Cruz Biotechnology, sc-816), 1:3,000 ENO2 (Cell Signaling Technology, 9536), 1:5,000 p21 (Cell Signaling Technology, 2947), and 1:2,000 ATRX (Cell Signaling Technology, 14820).