Generation and characterization of an antiserum reactive with a proteolytic processing site within rat procorticotrophin-releasing hormone

Castro, María G.; Rowe, Joanna; Murray, Christian; Tomašec, Peter; Shering, A.F.; Linton, Elizabeth A.; Ahmed, Irshad; Löwenstein, Pedro R.

doi:10.1016/0143-4179(95)90060-8

Cited by 4 publications

(6 citation statements)

References 21 publications

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“…The trasient transfections were performed as described previously (39). The cells were harvested for biochemistry or processed for immunofluorescence 24-48 h after transfection (15). Cell extracts for SDS and Western blotting were prepared as described previuosly (15).…”

Section: Antibody Productionmentioning

confidence: 99%

“…Rat tissue extract were prepared as described previously (15). Membranes were prepared as described (40) from pooled pregnant term myometrial samples taken from the upper border of the uterine incision during elective cesarian section of nine women aged 25-43 years.…”

Section: Rat Tissue Extracts and Human Myometrial Membranesmentioning

confidence: 99%

“…JRI, JR2 and JR3 peptides were iodinated by the solid-phase iodogen method (42). Samples were analysed using the radioimmunoassay methodology described previously (15). The rabbit anti-CRH receptor antibodies were used at a final dilution of 1/3,000 (JR2A), 1/15,000 (JR3A) or 1/30,000 (JR3B).…”

Section: Crh-receptor Radioimmunoassaymentioning

confidence: 99%

“…Tissue samples were lysed in 500 III SDS-sample buffer [0.07 M Tris pH 6.8, 2% (w/v) SDS, 10 % (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, 0.004% (w/v) bromophenol blue] containing I mM PM SF, then homogenized by 50 strokes in a Dounce homogenizer. Total cellular proteins were boiled for 10 min and were resolved using SDS as described previously (15). After electrophoresis, proteins were transferred onto 0.2 11m nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) using a semidry blotter (Hoefer UK, Newcastle-under-Lyme, UK).…”

Section: Sds and Western Blottingmentioning

confidence: 99%

“…Antibodies to the CRR-RI receptor should provide valuable research tools to study where and how CRH exerts its actions and targeting of CRR receptors within endocrine and neuronal cells. We have previously generated antibodies to synthetic peptide fragments corresponding to sequences within a protein which are capable of recognizing the full-size molecule, whether denatured or native, for example, for the CRR-binding protein (CRH-BP) (14) and the CRR precursor (15). Using the same approach, we have chosen three different peptide sequences within the CRH-RI molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on the surface of the native protein, for antibody production.…”

mentioning

confidence: 99%

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Corticotrophin‐Releasing Hormone Receptor Type 1: Generation and Characterization of Polyclonal Antipeptide Antibodies and their Localization in Pituitary Cells and Cortical Neurones in vitro

Castro

Morrison

Perone

et al. 1996

J Neuroendocrinology

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Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stress. CRH acts by first binding to specific receptors on the plasma membrane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH-R1). In this paper, we have chosen three different peptide sequences within the CRH-R1 molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on the surface of the native protein, for antibody production. Some of these fragments produced antipeptide antibodies of good titre which cross-reacted with the CRH-R1 receptor expressed in transiently transfected COS-7 cells and in tissue extracts from rat cerebellum, cortex, pituitary gland and human myometrium, both in Western blots and in liquid-phase radioimmunoassay. We used immunofluorescence techniques to localize the CRH receptor in transiently transfected COS-7 cells, primary cultures of rat anterior pituitary (AP) cells, the corticotroph-tumour cells AtT20 D16-16 and cortical neurons in primary culture. Our results indicate IR-CRH-R1 receptors have a punctate distribution on the plasma membrane of AP cells and AtT20 D16-16 cells. Whilst in AP cells their appearance is a fine punctate pattern, in AtT20 cells, they appear as large patches which could account for receptor clusters. Within primary cortical neurons, their distribution does not appear to be polarized. Our results suggest that distribution of CRH-R1 receptors within the different cell-types investigated depends not only on the amino acid sequence but also on cellular factors.

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Section: Antibody Productionmentioning

confidence: 99%

Section: Rat Tissue Extracts and Human Myometrial Membranesmentioning

confidence: 99%