Sstr2

Immunohistochemical analysis of pancreatic samples from 35 patients with CHI and 10 patients from the control group was carried out according to a standard method (27 (link)) using antibodies to: chromogranin A (rabbit polyclonal antibody, dilution 1:400, Diagnostic BioSystems, The Netherlands); insulin (mouse monoclonal antibody, clone K36aC10, 1:50 dilution, Diagnostic BioSystems, The Netherlands); Isl1 (rabbit polyclonal antibody, 1:1000 dilution, ThermoFisher, USA); Nkx 2.2 (rabbit monoclonal antibody, clone EPR14638, dilution 1:20, Abcam, UK); somatostatin (SST, rabbit polyclonal antibody, dilution 1:1000, Dako, Denmark); NeuroD1 (mouse monoclonal antibody, clone 3H8, dilution 1:1000, Novus Biologicals, USA); somatostatin receptor type 2 (SSTR2, rabbit monoclonal antibody, clone EP149, dilution 1:200, Abcam, UK); somatostatin receptor type 5 (SSTR5, rabbit monoclonal antibody, clone UMB4, dilution 1:100, Abcam, UK); type 1 dopamine receptor (DR1, rabbit polyclonal antibody, dilution 1:50, Novus Biologicals, USA); type 2 dopamine receptor (DR2, mouse monoclonal antibody, clone B10, dilution 1:100, Santa Cruz Biotechnology, USA); and type 5 dopamine receptor (DR5, rabbit polyclonal antibody, 1:100 dilution, ThermoFisher, USA). Micrographs were taken with a Leica DM4000 microscope and a Leica Aperio AT2 scanning microscope.

Cell lysates of PC3_VC and PC3_sgPTP1B were prepared by lysing the cells on ice for 30 min using freshly prepared buffer, which contained 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM sodium pyrophosphate, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 2 mM phenylmethylsulfonyl fluoride, and 1 mM EDTA. After centrifugation of the cell lysates at 14,000 rpm for 30 min at 4 °C, the protein extracts were loaded for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using Bolt 4–12% NuPAGE gels (Life Technologies, Carlsbad, CA, USA) and then blotted onto a nitrocellulose membrane using the Trans-Blot Turbo Transfer system (BIORAD, Hercules, CA, USA). After the membrane was incubated in the solution of primary antibodies against PTP1B (ProteinTech, Rosemont, IL, USA) and SSTR2 (Abcam, Cambridge, UK) at 4 °C for 16–18 h, it was washed and then incubated with horseradish-peroxidase-conjugated secondary antibodies at r.t. for 2 h. The blotting results were read using an Advansta ECL chemiluminescent detection system (San Jose, CA, USA). The loading control was β-actin (Santa Cruz Biotechnology, Dallas, TX, USA).

To examine the SSTR2 protein levels, tissue microarray analysis of 450 resected GTs from Seoul National University Hospital (SNUH-20040GC; SuperBioChips) (http://www.tissue-array.co.kr/main.html, accessed on 30 March 2019) was performed. The tissue array blocks contained up to 60 cores in an 8 array, for a total of 450 cases for immunohistochemical staining. Each core included more than 10% tumors and internal controls, such as nonneoplastic GM or IM. Immunohistochemical analysis was performed using a Leica Bond-max automated immunostainer (Leica Micosystems, Newcastle, UK) with antibodies against SSTR2 (Abcam, Cambridge, UK) or chromogranin A (ImmunoStar, Hudson, WI), an endocrine cell marker, as recommended by the manufacturer. SSTR2 staining patterns were scored as 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive), which correspond to negative (0) and positive (1, 2, and 3) groups, accordingly.