Secondary Antibody Incubation: Sections were incubated with Alexa Fluronanogold Fab fragment of goat anti rabbit IgG (Nanoprobes, NY) at a 1:25 dilution in 5% milk/5%serum in PBS for 2 hours and then rinsed with PBS, 45min. that are held together by tight junction complexes that effectively restrict the paracellular diffusion of solutes. Consequently the passage of polar substituents is mediated by transendothelial transport. Glucose is the primary energy substrate for mammalian brain and a constant supply is necessary to maintain cerebral activity. The original studies of Dick and colleagues were the first to demonstrate CETP-IN-3 the presence of the highly glycosylated (55 kDa) GLUT-1 glucose transporters in rat brain endothelial cells (Dick et al 1984). Earlier studies had demonstrated that this protein was also highly expressed in human erythrocytes where it mediated facilitated glucose transport and its presumed 2D structure is illustrated in Figure 1 (Blodgett and Carruthers 2008). Glucose transport across the BBB is not rate-limiting for cerebral metabolism under normal physiologic conditions; however under conditions of seizures, hypoxia, hypoxia-ischemia, chronic hypoglycemia it can become rate-limiting (see (Simpson et al 2007) for review. Analysis of the vectorial transport of glucose from blood to brain that encompasses the role of luminal to abluminal glucose transporters has also been long acknowledged as the appropriate mode to determine transport kinetics, however the molecular mechanism(s) underlying such transport have not been investigated (Cunningham et al 1986)(Choi et al 2001)(Simpson et al 2007). Open in a separate window Figure 1 GLUT-1 structure schematic (modified from Blodgett et al. 2008) to indicate primary antibody recognition sites. The structure of GLUT-1 glucose transporter showing the different regions of GLUT-1 from human erythrocytes presumed to be similar to the GLUT-1 at the blood-brain barrier. C-TER antibody recognizes the residues 472C492 at the C-terminus; the LOOP antibody recognizes the residues in the middle cytoplasmic loop from 231C246. H-SER antibody recognizes all the regions except the hydrophobic transmembrane domains. However, it may contain higher proportion of antibodies from the highly hydrophilic loop and C-terminus regions as shown for a similar whole sera antibody generated by Baldwin and colleagues (Davies et al. 1987). In a previous study, we demonstrated that the GLUT-1 glucose transporter exists in two distinct forms in the luminal and abluminal membranes of the bovine blood-brain barrier (Simpson et al 2001). Western blot analysis using a rabbit polyclonal antibody raised against the C-terminal 20 amino acids (C-TER) of GLUT-1 indicated a relative luminal: abluminal distribution of GLUT-1 of 1 1:5 that was entirely comparable to estimates obtained by electron microscopy studies of Pardridge and colleagues in rat, NAK-1 rabbit, and human endothelial cells (Cornford et al 1993; Cornford et al 1995; Farrell and Pardridge 1991; Gerhart et al 1989). However, when the same studies were performed using an antibody raised against the purified human erythrocyte GLUT-1 (H-SER), the ratio of luminal: abluminal GLUT-1 distribution was 1:1 (Sogin and Hinkle 1980) (Wheeler et al 1982). From these observations it was proposed that accessibility to the C-terminal epitope was compromised in the luminal membranes suggesting an alternative conformation. In this study, using the CETP-IN-3 GLUT-1 antibodies described above and immuno-electron microscopy, we confirmed that the distinct luminal and abluminal conformations observed by Western blotting are retained in rat brain microvessels in situ. Moreover, using a combination of 2D gel electrophoresis, Western blotting and alkaline phosphatases, we can now attribute these differing conformations to differential phosphorylation and not to different protein sequences resulting CETP-IN-3 from alternative splicing. MATERIALS AND METHODS Materials All chemicals and reagents were obtained from Sigma (St. Louis, MO) unless otherwise specified. TRIZOL reagent CETP-IN-3 was obtained from Invitrogen Life Technology (Cincinnati, OH). The source of all the GLUT-1 antibodies has been described previously (Simpson et al 2001). The C-terminal antibody (C-TER) was raised in rabbit against the amino acids 472C492 of the human GLUT-1 C-terminus which are common to rat and bovine CETP-IN-3 GLUT-1, the polyclonal loop antibody (LOOP) was raised in rabbit against residues 231C246 of the central cytoplasmic loop of human GLUT-1 (a gift from Dr. Anthony Carruthers). The antibody raised against.