Figure 8. Western analysis of GMP19 and GMP19-25

GMP19 (A) and GMP19-25 (B) stable cell lines were grown to confluence in 60 mm dishes and the synthesis of fusion protein induced with tetracycline. After three days of induction, the cells were prepared for analysis of integral membrane protein (Alkaline treated membranes), and lipid raft protein (Triton treated). Samples were subjected to SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes. Membrane were then washed, blocked, incubated with primary antibody (anti EGFP), washed again, and incubated with secondary antibody conjugated to alkaline phosphatase. The WesternBreeze chemiluminescent immunodetection system (Invitrogen) was used for immunodetection of EGFP fusion protein. These data clearly indicate that GMP19-25 (Figure 8B) does not associate with the cell membrane (remaining in the soluble fraction), while GMP19 not only is an integral membrane protein but also associates with lipid rafts (Figure 8A). MW refers to the molecular weight standards. S refers to the soluble protein fraction and P refers to the pellet (insoluble) protein fraction.