Prdx1

Protein lysates were prepared from olfactory bulbs and hippocampi of 5 months old WT and KO female mice using RIPA buffer with protease inhibitors followed by sonication by ultrasound and clearing via centrifugation at maximum speed using Eppendorf tabletop centrifuge. Protein concentration was quantified using BCA reagent (Thermo Fisher Scientific, Rockford, IL). Protein extracts (40 μg/well) were subjected to Western immunoblot analysis. We used antibodies against the following proteins: PRDX1 (Sigma), ACC1, pACC1, AMPKα, pAMPKα, NGFR, TrkB, Bcl-xL, Rack1 (all from Cell Signaling Technology), Calretinin (Millipore), LC3 (Abcam). The band intensity was determined using ImageJ software, normalized to loading control (β-actin). The data are presented as a relative intensity of WT bands over KO bands. Phospho-index was determined as a ratio of phosphorylated/total protein intensities.

Lymph nodes from 4 patients with reactive follicular hyperplasia and tissue sections from gastric mucosa and tumors of the thigh, neck and thyroid gland from 4 patients with BL were evaluated. All individuals gave informed consent and the study was approved by the Institute of Hematology and Transfusion Medicine Ethics Committee. Tissue biopsies of the patients were histopathologically examined to establish a diagnosis according to 2008 WHO classification. Tissue biopsies were fixed in 10% formalin, routinely processed and stained with hematoxylin and eosin. Immunohistochemistry was performed using the following antibodies: PRDX1 (Sigma-Aldrich, dilution 1:150, pH = 9.0) and PRDX2 (GeneTex, clone EPR5154, dilution 1:200, pH = 9.0). Staining was performed according to the manufacturer's instructions. The EnVision System (Dako) was used for detection. Positive controls included human tonsils (PRDX1) and human prostatic hyperplasia (PRDX2). The negative (isotype) controls were generated with ready to use FLEX Negative Control Mouse (cocktail of mouse IgG1, IgG2a, IgG2b, IgG3 and IgM; code No IR750; Dako). Samples were reviewed for the expression of lymphoid cells by MPS. Appropriate cellular localization for immunostaining was membrane and cytoplasmic for PRDX1 and PRDX2. All photographs were taken using DP72 Olympus BX63 microscope camera (Olympus, Japan).