Anti ip6k1

Protein lysates were prepared using the Qproteome Mammalian Protein Prep Kit (Qiagen) and protein concentrations were determined using the Bradford assay (Bio‐Rad, Hercules, CA, www.bio-rad.com). Protein samples (20 µg) were prepared in Laemmli sample buffer (Bio‐Rad) containing 5% β‐mercaptoethanol, denatured at 95°C for 5 minutes, electrophoresed on NuPAGE 10% Bis‐Tris gels using 1× NuPAGE MES SDS Running Buffer (Thermo Fisher Scientific), and then transferred to 0.45 μm nitrocellulose membranes in 1× NuPAGE transfer buffer containing 10% methanol. Membranes were washed with Tris‐buffered saline (TBS) for 5 minutes, incubated in TBS with 0.1% Tween‐20 (TBST) and Odyssey^® blocking buffer (LI‐COR Biosciences, Lincoln, NE, www.licor.com) overnight at 4°C, washed an additional 3× in TBST, and then incubated with anti‐IP6K1(1:500, GeneTex, Irvine, CA, www.genetex.com), anti‐p53, anti‐MDM2, anti‐p16, and anti‐GAPDH (1:200, Santa Cruz Biotechnology Inc., Dallas TX, www.scbt.com) antibodies in Odyssey^® blocking buffer for 2 hours at room temperature with gentle agitation. Membranes were washed 5× in TBST and probed with a Infrared (IR) dye‐labeled secondary antibody at a 1:10,000 dilution in Odyssey^® blocking buffer for 1 hour. Blots were scanned using Odyssey^® infrared image system (LI‐COR Biosciences).