Cell lysis and immunoblotting was essentially performed as described previously [42 (link)]. In brief, cells were washed once with ice-cold PBS and lysed with PhosphoSafe lysis buffer (Merck Millipore, Darmstadt, Germany). Following clearance of the lysates by centrifugation, their total protein concentrations were determined with the DC Protein Assay (BioRad). Equal amounts of proteins were fractionated by PAGE on mini-PROTEAN TGX any-kD precast gels, or TGX Stain-Free FastCast gels (BioRad) and blotted to PVDF membranes. Membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies (CgA: Monoclonal Mouse Anti-Human Chromogranin A, clone DAK-A3, Dako, Glostrup, Denmark; SYP: Anti-Synaptophysin, Dako; SSTR2: Anti-Somatostatin Receptor 2 antibody [UMB1]-C-terminal #ab134152, Abcam, Cambridge, UK) overnight at 4 °C. After washing and incubation with horseradish peroxidase-linked secondary antibodies, chemoluminescent detection of proteins was done on a ChemiDoc XRS imaging system (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to those for the housekeeping genes GAPDH or HSP90.
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