Anti arf

Collected cells were washed with cold PBS and lysed in ice-cold buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40] supplemented with complete protease inhibitors (Sigma-Aldrich) on ice for 10 min. The samples were centrifuged at 13,000rpm for 10 min. The lysates were aliquoted into two tubes and incubated with either the designated antibody or an appropriate IgG control for 16 hours at 4 °C. Protein A/G agarose (Invitrogen) was used to precipitate antibody-protein complexes.
For Western blotting, anti-CD24 (Santa Cruz Biotechnology, 1:1000), anti-p27 (Cell Signaling, 1:1000), anti-p21 (Cell Signaling, 1:1000), anti-p53 (Santa Cruz Biotechnology, 1:2000), anti-Lamin (Santa Cruz Biotechnology, 1:5000), anti-H1.5 (Abcam, 1:5000), anti-Cyclin B1 (Santa Cruz Biotechnology, 1:3000), anti-GFP (Cell Signaling, 1:2000), anti-NPM1 (Abcam, 1:3000), anti-HA (Sigma-Aldrich, 1:3000), anti-Myc (Abcam, 1:3000), anti-ARF (Santa Cruz Biotechnology, 1:1000), anti-MDM2 (Santa Cruz Biotechnology, 1:1000), and anti-β-actin (Sigma, 1:5000) antibodies were used as primary antibodies. The membranes were incubated for 1 hour at room temperature in 0.25% nonfat milk with a 1:3,000–5,000 dilution of either anti-rabbit or anti-mouse IgG HRP-linked secondary antibody (Cell Signaling).