Glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) a GPI-anchored endothelial cell protein binds lipoprotein lipase (LPL) and transports it in to the lumen of capillaries where it hydrolyzes triglycerides in lipoproteins. be possible to define GPIHBP1 expression patterns with radiolabeled GPIHBP1-specific antibodies and positron emission tomography (PET) scanning. In expression expression was also high but the lung was an exception (very high expression and extremely low expression). Despite low transcript levels Olmesartan however LPL protein was readily detectable in the lung suggesting that some of that LPL originates elsewhere and then is usually captured by GPIHBP1 in the lung. In support of this concept lung LPL levels were significantly lower in knock-out mice (gene. The latter mice express substantial amounts of human LPL activity in skeletal muscle but the levels are undetectable in adipose tissue (11). Mice were fed a chow diet and housed in a barrier facility with a 12-h light-dark cycle. All studies were approved by the UCLA Animal Research Committee. Antibodies For the GPIHBP1 biodistribution studies we used a pair of rat monoclonal antibodies (mAbs) against mouse GPIHBP1 11 and 2A8 (12). Control antibodies included a rat monoclonal antibody of the same isotype 16 a hamster monoclonal antibody against CD31 2 (Millipore Billerica MA); and a Olmesartan hamster monoclonal antibody against EMR 30000000 (Abcam Cambridge MA). Immunohistochemistry To detect GPIHBP1 in mouse tissues 8 thick frozen sections were prepared and processed for immunohistochemistry as described (7). Alexa Fluor 555-labeled mAb11A12 (3 μg/ml) was used to detect GPIHBP1. Endothelial cells were identified with the hamster anti-CD31 monoclonal antibody (1:200) and Alexa Fluor 488-labeled goat anti-hamster IgG (1:200). Extracellular matrix was identified with a rabbit anti-collagen IV antibody (1:1000) and an Alexa Fluor 488-labeled donkey anti-rabbit IgG (1:200). Images were obtained with an Axiovert 200 MOT microscope equipped with an Apotome (Zeiss Germany) or by confocal fluorescence microscopy with a Leica SP2 1P-FCS microscope (Heidelberg Germany). Western Blots Tissue extracts were prepared in radioimmunoprecipitation assay buffer (RIPA: 1× PBS 1 Nonidet P-40 0.5% sodium deoxycholate and 0.1% SDS) containing complete mini EDTA-free protease inhibitors (Roche Applied Science). Extracts were size-fractionated on 12% polyacrylamide BisTris gels (Invitrogen) and the separated proteins were transferred to nitrocellulose for Western blotting. Antibody dilutions were 1:200 for a goat antibody against lamin A/C (sc-6215 Santa Cruz Biotechnology); 1:1000 for mAb11A12 (12); and 1:1000 for a goat antibody against a recombinant mouse LPL fragment (13). The binding of IR-coupled secondary antibodies was detected and quantified with an Odyssey infrared imaging scanner (Li-Cor Lincoln NE). Quantitative RT-PCR Total RNA was prepared from mouse tissues with TriReagent (Sigma) treated with DNase I (Ambion Austin TX) and reversed transcribed into cDNA with a mixture of random primers and oligo(dT) and Superscript III (Invitrogen). Primers 5′-AGCAGGGACAGAGCACCTCT-3′ and 5′-AGACGAGCGTGATGCAGAAG-3′ were used to amplify the mouse cDNA; primers 5′-AGGTGGACATCGGAGAACTG-3′ and 5′-TCCCTAGCACAGAAGATGACC-3′ were used to amplify mouse cDNA; primers 5′-TAGCTGGTCAGACTGGTGGA-3′ and 5′-TTCACAAATACCGCAGGTG-3′ were used to amplify human cDNA; and primers 5′-TGGTGCTTGTCTCACTGACC-3′ and 5′-TATGTTCGGCTTCCCATTCT-3′ were used to amplify mouse β2-microglobulin cDNA. Quantitative PCR was performed on 50 ng of cDNA 200 nm of each Olmesartan primer and 10 μl of SYBR Green PCR grasp mix (Qiagen Valencia CA). PCR were performed in triplicate on a 7900HT Fast Real Time PCR system (Applied Biosystems Foster City CA). Gene expression levels Olmesartan normalized to β2-microglobulin were calculated by the comparative method. Dimension of LPL in Tissue Mice overnight were fasted. After adding Nrp2 meals towards the cages for 1 h the mice had been fasted for 4 h before getting euthanized. Tissues (100 Olmesartan mg) was homogenized using a Fisher Scientific PowerGen 125 in 1.0 ml of lysis solution (13). Examples had been centrifuged at 20 0 × for 30 min at 4 °C. The supernatant fractions had been kept and gathered at ?80 °C. LPL amounts in these examples.