D5j4e

NOPE expression was analyzed for correlation with immunohistochemical markers including integrin alphaV expression (ITGAV) and Indoleamine 2,3-dioxygenase (IDO) expression on tumor infiltrating lymphocytes (TILs) and tumor cells. Immunohistochemistry (IHC) was performed on TMA slides using the monoclonal rabbit anti—ITGAV antibody (ab150361; dilution 1:300; Abcam, UK) and the monoclonal rabbit IgG anti—IDO antibody (D5J4E; dilution 1:400; Cell Signaling Technology, USA). Both staining procedures are described elsewhere in detail^{17 (link),18 (link)}.

Immunohistochemistry (IHC) was performed on TMA slides. For IDO the rabbit IgG monoclonal antibody (D5J4E; dilution 1 : 400; Cell Signaling Technology, USA) and for CD3 the rabbit monoclonal antibody (SP7; dilution 1 : 50; Thermo Fisher Scientific, USA) were used. All immunohistochemical stainings were performed using the Leica BOND-MAX stainer (Leica Biosystems, Germany) according to the protocol of the manufacturers. The evaluation of immunohistochemical expression was assessed manually by two pathologists independently (PL and HL). Discrepant results were resolved by consensus review.

IDO1 protein expression was analyzed by means of a rabbit monoclonal anti-human IDO1 antibody (D5J4E™, Cell Signaling Technology), whereas SHP-2 protein level was detected by the use of a mouse monoclonal anti-SH-PTP2 antibody (clone B-1, Santa Cruz Biotechnology). Moreover, a mouse monoclonal anti-β-tubulin antibody (clone AA2, Sigma-Aldrich) was used as normalizer. SKOV-3 cells were lysed in Laemmli buffer containing 2-mercaptoethanol and used for sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). After incubation with the specific horseradish peroxidase-conjugated secondary antibodies (Sigma-Aldrich) and the addition of Clarity™ Western ECL (Enhanced ChemiLuminescence, Bio-Rad Laboratories) substrate, signals were detected by the use of ChemiDoc™ MP Imaging System (Bio-Rad Laboratories). Protein expression was quantified by densitometric analysis, using ImageLab Software (Bio-Rad Laboratories), as previously described (14 (link)). The enzymatic activity of IDO1 was evaluated in cell culture supernatants in terms of the ability of the enzyme to metabolize tryptophan into kynurenine, by HPLC analysis as previously described (20 (link)).