Ab59539

The validation of proteomics data was performed by WB for two proteins GAPDH and PRDX2 on RBC samples from Cohort II (Table 2) and Cohort III (Table S1), respectively. RBC samples were 1:100 diluted with 100 mM of N-ethylmaleimide (NEM, crystalline, ≥98% HPLC, Sigma Aldrich, Darmstadt, Germany) in PBS buffer for 10 min at 4 °C to prevent exogenous-induced oxidation. Samples (500 µL) were lysed in 1:1 double distilled (dd) H₂O with protease inhibitors cocktail 1:100 (P8340, Sigma Aldrich, Darmstadt, Germany), centrifuged at 9279× g for 10 min at 4 °C (Centrifuge 5417, Eppendorf, Hamburg, Germany), and the cytoplasmic supernatants were recovered for protein quantification as above described.
To keep protein disulfide bonds formation in an oligomerization state, samples were separated (70 ug/lane) by non-reducing 4–12% SDS-PAGE mini gels followed by WB analysis using 1:2000 rabbit polyclonal anti-GAPDH antibody (ab9485, abcam, Cambridge, UK), 1:500 mouse monoclonal anti-GAPDH-SO₃ antibody (4A1) (Abfrontier, South Korea), 1:20,000 rabbit anti-PRDX2 antibody (ab59539, abcam, Cambridge, UK), or 1:3000 rabbit anti-PRDX-SO_2/3 antibody (ab16830, abcam, Cambridge, UK) to investigate the different redox oligomeric states of GAPDH and PRDX2, respectively, as described [20 (link),21 (link)].

Western blot analysis was conducted to confirm the results of proteomic analysis. The heart tissues collected at 6 weeks of age were homogenized in lysis buffer (T-PER reagent; Thermo Fisher Scientific, Waltham, MA). Samples (n = 6 per group) containing 12 μg cardiac proteins were separated by 12% SDS-PAGE and electroblotted onto polyvinylidenedifluoride (PVDF) membranes. The membranes were incubated with a rabbit polyclonal antibody to peroxiredoxin 2 (PRDX2) (ab59539; Abcam, Cambridge, United Kingdom) at 1:2,000 dilution. A rabbit monoclonal antibody to GAPDH at 1:2,000 dilution (14C10; Cell Signal Technology, Danvers, MA) was used as a loading control. The protein bands were visualized by ECL plus Western blotting detection system (GE Healthcare) and Quantity One v3.0 software (Bio-Rad Laboratories) was used to quantitate the band intensities. To prevent the detection as a saturated signal, we confirmed the antibody dilution concentration and exposure time of the CCD camera in the preliminary experiments. The expression level of PRDX2 was normalized relative to the level of GAPDH protein in the same tissue sample.

HCC and para-tumor tissues were fixed, dehydrated, cleared, immersed in wax, embedded, and sectioned. Paraffin sections were then subjected to gradient ethanol dehydration and antigen retrieval using sodium citrate. After blocking, the sections were incubated overnight at 4°C with primary antibodies against PRDX2 (Abcam, ab59539, 1:50). Then, HRP-labeled secondary antibodies (1:200, Abcam) were applied and incubated at 37°C for 30 min. The sections were subsequently stained with diamino-benzidine (DAB, Maxim Biotech, Fuzhou, China) and hematoxylin (Solarbio, China; cat. no. H8070), followed by differentiation, hydration, and making them transparent. Finally, the slides were sealed, and the results were observed under a microscope (Nikon, Tokyo, Japan).