5HT Serotonins

Prem N. Yadav, Wesley K. Kroeze, Martilias S. Farrell and Bryan L. Roth. ANTAGONIST FUNCTIONAL SELECTIVITY: 5-HT2A SEROTONIN RECEPTOR ANTAGONISTS DIFFERENTIALLY REGULATE 5-HT2A RECEPTOR PROTEIN LEVEL IN VIVO. DOI:10.1124/jpet.111.18378.

...For 5-HT2A receptor immunoblotting, frontal cortex crude membrane preparations were resuspended in cold lysis buffer (50 mM Tris-Cl, pH 7.4; 150 mM NaCl, 10% glycerol, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, plus protease inhibitor cocktail (Roche Diagnostics) and incubated on ice for 1 hr. Detergent-soluble proteins were collected after 20 min of centrifugation at 4 0 C and 12,500g, and 500 μg (1.0 mg/ml) of protein was incubated with 25-30 μl (packed volume) of wheat germ agglutinin (WGA)-conjugated agarose beads (Vector laboratories, Inc. CA) for a minimum of 2 hrs at 4 0 C on a rotary mixer. After three washes with 500 μl of lysis buffer, WGA-bound proteins were eluted with 40 μl of 1X SDS-sample buffer, and whole eluates were resolved by SDS-PAGE, followed by electrotransfer of protein from gel to PVDF membrane. Membranes were blocked for one hr using buffer containing 3% non-fat dry milk, 1 % BSA and 5% glycerol in TBST. Rabbit polyclonal anti-5-HT2A receptor antibody (Neuromics, Edina, MN) was used at a 1:500 dilution for 12-18 hrs at 4 0 C for measurement of 5-HT2A receptor immunoreactivity. For loading control, same membrane was stripped and reprobed with anti-transferrin receptor mouse monoclonal antibody (Invitrogen Corp., CA). We have previously verified the specificity of the anti-5-HT2A receptor antibody using 5-HT2A WT and KO mice cortex tissue by immonoblotting (Magalhaes et al., 2010)...

Prem N Yadav, Atheir I Abbas, Martilias S Farrell, Vincent Setola, Noah Sciaky, Xi-Ping Huang, Wesley K Kroeze, LaTasha K Crawford, David A Piel, Michael J Keiser, John J Irwin, Brian K Shoichet, Evan S Deneris, Jay Gingrich, Sheryl G Beck and Bryan L Roth. The Presynaptic Component of the Serotonergic System is Required for Clozapine's Efficacy. Neuropsychopharmacology , (3 November 2010) | doi:10.1038/npp.2010.195.

...Rabbit polyclonal anti-5-HT2A receptor antibody (Neuromics, Edina, MN) was used in a 1 : 500 dilution for 12 h at 4 °C...

Cullen L. Schmid and Laura M. Bohn. Serotonin, But Not N-Methyltryptamines, Activates the Serotonin 2A Receptor Via a β-Arrestin2/Src/Akt Signaling Complex In Vivo. J. Neurosci., Oct 2010; 30: 13513 - 13524 ; doi:10.1523/JNEUROSCI.1665-10.2010.

...antibody to the N terminus of the 5-HT2AR (1:500) was from Neuromics (Magalhaes et al., 2010); the c-Myc monoclonal antibody (1...rabbit antibody against the N terminus of the 5-HT2AR (1:133; Neuromics) at 4 complexes were immobilized on Protein A Sepharose 4...

Ana C Magalhaes,Kevin D Holmes,Lianne B Dale,Laetitia Comps-Agrar,Dennis Lee,Prem N Yadav,Linsay Drysdale,Michael O Poulter,Bryan L Roth,Jean-Philippe Pin,Hymie Anisman& Stephen S G Ferguson.CRF receptor 1 regulates anxiety behavior via sensitization of 5-HT2 receptor signaling. Nature Neuroscience. doi:10.1038/nn.2529. Published online11 April 2010.

...Brains were harvested, placed in 4% paraformaldehyde in 1× PBS at 4 °C for 4 h, moved to 30% sucrose (wt/vol) in 1× PBS until they sank, brains were then frozen on dry ice and stored at −80 °C. Brain sections (30 μm) were prepared and free-floating sections in 1× PBS (one per well in a 24-well plate) were permeabilized with 0.4% Triton X-100 in 1× PBS for 1 h. 1× PBS /0.4% Triton X-100 containing 0.1% glycine (wt/vol), 0.1% lysine (wt/vol), 1% BSA (wt/vol) and 1% normal donkey serum (wt/vol). Primary antibodies (rabbit polyclonal antibody to 5-HT_2A, Neuromics cat # RA24288; goat polyclonal antibody to CRFR1, Abcam cat # ab59023) were incubated in blocking buffer for 72 h at 4 °C. Sections were then washed five times in 1× PBS/0.4% Triton X-100 (10 min each). Hoechst (2–5 μg ml⁻¹) and secondary antibodies (donkey Alexa Fluor 555 conjugated anti-goat (1:500) and donkey Alexa Fluor 488 conjugated anti-rabbit antibodies (1:500); Invitrogen) were diluted in blocking buffer and slices were incubated for 1 h at 20–23 °C. Sections were washed five times in 1× PBS/0.4% Triton X-100 (10 min each). Sections were mounted on slides and visualized by Zeiss LSM-510 META multophoton laser-scanning microscope with a Zeiss 25× NA 1.2 oil immersion lens and appropriate filters... 

Cullen L. Schmid, Kirsten M. Raehal, and Laura M. Bohn. Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo. Published online on January 14, 2008, 10.1073/pnas.0708862105

... Neurons were incubated with 5-HT2AR polyclonal rabbit (1:100; Neuromics)...

Deborah Conte, Susan A Deuchars, Ferenc Erdelyi, Gabor Szabo, Jim Deuchars. Serotonin immunoreactive terminals closely appose GABAergic neurones in autonomic regions of the spinal cord. University College London 2006 (2006) Proc Physiol Soc 3 PC77

...Thoracic spinal cord was sectioned at 50µm on a vibratome (Leica). Serotoninergic terminals were localised using rabbit anti-5-HT (1:500, Neuromics Inc, USA) and visualised with donkey anti-rabbit Alexa555 (1:1000, Invitrogen, UK)...

Małgorzata Lehner, Ewa Taracha, Anna Skórzewska, Piotr Maciejak, Aleksandra Wisłowska-Stanek, Małgorzata Zienowicz, Janusz Szyndler, Andrzej Bidziński and Adam Płaźnik. Behavioral, immunocytochemical and biochemical studies in rats differing in their sensitivity to pain. doi:10.1016/j.bbr.2006.03.044  

...slide-mounted brain sections were incubated in rabbit polyclonal serotonin antiserum diluted at 1:100 (Neuromics) in temperature 4–8 °C for 24 ...

Naohisa Tsujino, Yasushi Nakatani, Yoshinari Seki, Akane Nakasato, Michiko Nakamura, Michiya Sugawara and Hideho Arita. Abnormality of circadian rhythm accompanied by an increase in frontal cortex serotonin in animal model of autism. doi:10.1016/j.neures.2006.10.018  

Rong-Jian Liu, Evelyn K. Lambe and George K. Aghajanian. Somatodendritic autoreceptor regulation of serotonergic neurons: dependence on l-tryptophan and tryptophan hydroxylase-activating kinases. European Journal of Neuroscience, Vol. 21, pp. 945–958, 2005

Syann Lee, Christine L. Walker,Barbara Karten, Sharee L. Kuny, Alysa A. Tennese, Megan A. O'Neill and Rachel Wevrick.  Essential role for the Prader–Willi syndrome protein necdin in axonal outgrowth. Human Molecular Genetics 2005 14(5):627-637