Pd l1 e1l3n

Three million pCMV Vector or miR-200c transfected SKOV3 cells were collected and lysed in RIPA lysis buffer. Thirty micrograms of protein of each sample was loaded on 10% polyacrylamide gels and run for 1.5 h at 120 V. The following antibodies were used: PD-L1 (E1L3N) (Cell Signaling Technology, Danvers, MA, USA; Cat. n.13684), β-catenin (E-5) (Santa Cruz Biotechnology, Inc., Heidelberg, Germany; Cat. n.sc-7963), and c-Myc (D84C12) (Cell signaling; Cat. n.5605). β-actin (C4) (Santa Cruz; Cat. n.sc-47778) and Lamin B1 (C-20) (Santa Cruz; Cat. n.sc-6216) were used to ensure equal protein loading. HRP conjugated anti-rabbit (SIGMA; Cat. n.A 6154) and anti-mouse (ADVANSTA; Cat. n.R-05071-500) secondary antibodies were used, (1:5000 in 2% BSA) for 30 min. The chemiluminescent signal was detected using WesternBright^® ECL (ADVANSTA, San Jose, CA, USA; Cat. n.K-12045-D20). Densitometry analysis was performed with ImageJ Software (v. 10.2). Immunoblots were repeated three times with the same lysates and with protein lysates derived from three different treatments of olaparib and irradiation. Additional details of immunoblotting conditions can be found in Supplementary Materials and Methods.

Immunohistochemistry for all antibodies was performed using 3 μm thick slides and standard protocols on the automated IHC staining system Discovery XT (Roche/Ventana, Tucson, Arizona, USA). The following antibodies were used: CD3 (A0452, dilution 1:500, DAKO, Glostrup, Denmark), CD8 (clone C8/144B, dilution 1:100, DAKO, Glostrup, Denmark), PD-1 (clone NAT105; dilution 1:50; Abcam, Cambridge, United Kingdom), PD-L1 (E1L3N; dilution 1:200; Cell Signaling, Boston, U.S.A.), FOXP3 (clone 236A/E7; dilution 1:100; eBioscience, San Diego, U.S.A.) Slides were counterstained with hematoxylin and mounted.

Two µm thick unstained slides from the TMA were stained on a Leica Bond stainer (Leica Biosystems, Wetzlar, Germany) with antibodies against synaptophysin (DAK-SYNAP, Agilent Dako, Santa Clara, CA, USA), chromogranin A (LK2H10, Zytomed, Berlin, Germany), serotonin (5HT-209, Agilent Dako, Santa Clara, CA, USA), PD-L1 (E1L3N, Cell Signaling, Boston, MA, USA), CD3 (F7.2.38, Agilent Dako, Santa Clara, CA, USA), CD20 (L26, Agilent Dako, Santa Clara, CA, USA), and CDX-2 (DAK-CDX2, Agilent Dako, Santa Clara, CA, USA). Specific external control tissues for each antibody were deposited as a staining reference on each slide of the TMA.
Two µm thick unstained full-block slides from FFPE blocks were stained with a Ki-67 antibody (MIB-1, Agilent Dako, Santa Clara, CA, USA) and an SSTR2a antibody (polyclonal, Zytomed, Berlin, Germany). For Ki-67, the proliferation zone of the basal epithelium of the jejunoileal mucosa served as an internal staining control on the respective slide. For SSTR2a normal endocrine pancreatic islets served as staining control.

IHC staining was performed using the Ventana Discovery System (Ventana Medical System, Oro Valley, AZ, USA) [48 (link)]. Briefly, slides were incubated at room temperature with primary antibodies: anti-CDK19 polyclonal rabbit (HPA007053, Sigma, St. Louis, MO, USA), pSTAT1 (D4X3C, Cell Signaling, Danvers, MA, USA), PD-L1 (E1L3N, Cell Signaling, Danvers, MA, USA), and detected with the ultraView Universal DAB Detection Kit (Ventana Medical System, Tucson, AZ, USA).

For protein extraction, lung tissue samples were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) added with an inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Lung tissue samples were homogenized using SpeedMill PLUS (Analytik Jena, Jena, Germany) and innuSPEED lysis Tube P (Analytik Jena). After homogenization samples were centrifuged (5 min, 3000 rpm, 4°C), supernatants were collected and incubated on ice for 45 min followed by centrifugation (5 min, 3000 rpm, 4°C; 45 min, full speed, 4°C). Protein concentration was determined using Bradford Assay (Protein Assay Dye Reagent Concentration, Bio-Rad, Munich, Germany). Western blot analysis to detect Phospho-STAT1 Tyr701 (1/1000, cell signaling, Danvers, MA, USA), PD-L1 E1L3N (1/1000, cell signaling) and b-Actin (1/500, sc-1616, Santa Cruz, TX, USA) was performed as described in [39 (link)] with 50 μg of total protein. Quantification of total p-Tyr-STAT1 and PD-L1 was performed using AlphaView Software for FluorChem Systems (Biozym Scientific, Oldendorf, Germany).