Western blot using the affinity purified anti-PTEN-P1 antibody shows detection of endogenous PTEN-P1 in whole cell lysates from human derived cell lines HeLa (lane 4), HEK293 (lane 5) and MCF7 (lane 6). The band at ~55 kDa (arrowhead) corresponds to PTEN-P1. Lanes 1-3 were show the results of staining after the antibody was first pre-incubated with the immunizing peptide. The identity of lower molecular weight bands in lane 4 is unknown. Briefly, each lane contains approximately 35 µg of lysate. Primary antibody was used at a 1:500 dilution in 5% BLOTTO in PBS reacted overnight at 4°C. The membrane was washed and reacted with a 1:10,000 dilution of conjugated Gt-a-Rabbit IgG [H&L] MX for 45 min at room temperature (800 nm channel, green). Molecular weight estimation was made by comparison to prestained MW markers in lane M (700 nm channel, red). Fluorescence image was captured using the Odyssey Infrared Imaging System developed by LI-COR. Other detection systems will yield similar results.