Anti beclin1

Primary antibodies anti-Beclin1 and anti-LC3 (catalogue numbers: GTX113039 and GTX48634 respectively; GeneTex Inc., Irvine, CA), anti-ULK1 (catalogue number: EPR4885(2); Abcam, Cambridge, UK), and anti-Atg7 (catalogue number: DF6130; Affinity Biologicals Inc, Ontario, Canada) were used with corresponding secondary antibodies (Dako, Ely, UK).

Cisplatin, amifostine (AMF), adenosine, and N6-(2′-hydroxyethyl) adenosine (HEA), as well as other reagents and solvents, were obtained from Sigma-Aldrich (St. Louis, MO, USA). Commercial assay kits for BUN and CRE were provided by HUMAN Diagnostics Worldwide (Wiesbaden, Germany). Mouse TNF-α, IL-1β, and IL-6 ELISA Max™ Set Deluxe Kits were purchased from BioLegend Inc. (San Diego, CA, USA). Primary antibodies for Western blots against anti-COX-2, anti-p-JNK, anti-catalase, anti-GPx, anti-SOD, anti-AMPK, anti-Sirt1, anti-TLR4, anti-P62, anti-Beclin-1, anti-cleaved PARP, and anti-cleaved caspase were purchased from GeneTex (San Antonio, TX, USA). Anti-OAT1 and anti-OAT3 were purchased from ABclonal (MA, USA). Antibodies against anti-JNK, anti-p-ERK, anti-ERK, anti-p-p38, anti-p-IκBα, and anti-LC3B were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against anti-iNOS, anti-NF-κB, anti-IκBα, anti-p38, anti-HO-1, anti-Nrf-2, and β-actin were purchased from Abcam (Cambridge, UK). Protein assay kits (Bio-Rad Laboratories Ltd., Watford, Herts, UK) were obtained as indicated.

The Tissue Micro Array (TMA), provided by the Tissue Bank of the National Center for Tumor Diseases (NCT, Heidelberg, Germany), consists of 10 spots for normal mucosa, 18 spots for adenoma and 49 spots for adenocarcinoma. Each spot represents a single patient. Paraffin embedded sections were stained with the respective antibody, using NovoLink Polymer detection System (Leica Microsystems, Wetzlar, Germany) according to the manufacturer’s instructions. Negative controls were generated by omitting the primary antibody. The Immunoreactivity Score was calculated by two independent investigators as described before [48 (link)], with separate scores for quantity (0–10% = 1, 11–50% = 2, 51–80% = 3, 81–100% = 4) and quality (unstained = 0, weak = 1, moderate = 2, strong = 3). The following antibodies were used for detection: anti-Atg7 (Cell Signaling Technology, Danvers, MA, USA, No. 8558), anti-p62 (Becton Dickinson, Heidelberg, Germany, No. 610832) and anti-Beclin-1 (GeneTEX, Irvine, CA, USA, No. gtx37770) and anti-LC3b (Cell Signaling Technology, No. 3868). For analysis, scoring was done manually by two investigators. The usage of patient tissue for research purposes was approved by the local ethics committee of the University Hospital of Heidelberg (S-206/2005). All analyses were done anonymously and written informed content was obtained from all donors.