Technical Reference #1787

1787.

Pasireotide and Octreotide Stimulate Distinct Patterns of sst2A Somatostatin Receptor Phosphorylation Florian Poll; Diana Lehmann; Susann Illing; Mihaela Ginj; Stefan Jacobs; Amelie Lupp; Ralf Stumm; Stefan Schulz, Friedrich-Schiller University, Molecular Endocrinology, 24(1787), (2010)
Link To Paper

Materials & Methods:
Reagents and antibodies SOM230 and octreotide were provided by Herbert Schmid (Novartis Basel Switzerland). L-779976 was a gift from Susan Rohrer (Merck Rahway NJ). BIM-23627 was provided by Michael Culler (IPSEN Milford MA). Somatostatin (SS-14) was obtained from Bachem (Weil am Rhein Germany). The phosphorylation-independent rabbit monoclonal anti-sst2A antibody (UMB-1) and the rabbit polyclonal anti-T7 antibody were generated and extensively characterized as previously described (24 38 39). Phosphosite-specific polyclonal antisst 2A antisera were generated against the following sequence that contains four phosphorylated threonine residues: RLNE(pT)(pT)E(pT)QR(pT)LLNG. This sequence corresponds to amino acids 349–363 of the rat sst2A receptor. The peptide was purified and coupled to keyhole limpet hemocyanin. The conjugate was mixed 1:1 with Freund’s adjuvant and injected into four rabbits (0479 0490 0521 and 0522) for antisera production. Animals were injected at 4-wk intervals and serum was obtained 2 wk after immunizations beginning with the second injection. The specificity of the antisera was initially tested using Dot blot analysis. For subsequent analysis antibodies were affinity purified against their immunizing peptide as well as against the peptides RLNE(pT)(pT)ETQRTLLNG (peptide 1) and RLNETTE(pT)QR(pT)LLNG (peptide 2) using the SulfoLink kit (Thermo Scientific Rockford IL). Antibodies (4465–4467) to RKIP were generated against the peptide CFQAEWDDSVPKLHDQLAGK which corresponds to amino acids 168–187 of the rat RKIP.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
INS1 cells; GH3 cells; HEK293 cells