Quantitative Western Blotting of PTEN

Whole cell extracts were prepared as previously described [22 (link)]. Protein (30 μg) was analyzed on a Novex^® 4–12% Tris-Bis gradient polyacrylamide gel and immunoblot analysis performed as described previously [32 (link)]. Primary antibodies used were β-actin (1:200) produced in mouse (C4:Santa Cruz Biotechnologies Inc.) and PTEN (1:1000) produced in rabbit (D4.3:Cell Signaling). After incubation in primary antibody overnight, the blot was washed as previously described [32 (link)] and incubated first in secondary anti-mouse IgG-HRP (1:1000) produced in goat (Santa Cruz Biotechnologies) and then with anti-rabbit IgG-HRP (1:1000) produced in goat (Cell Signaling) for 1 hour at room temperature. After washing bands were developed using the chemiluminescent reagents from Western Lightning^® (Perkin Elmer, Boston, MA). Western blots were exposed and bands quantitated using the ODYSSEY^® Fc Imaging System (LI-COR Biosciences, Lincoln, NE).