Fig. 4.

The CSN5:Prdx2 interaction upon H₂O₂ level elevation was confirmed by proximity ligation, but not with co-immunoprecipitation (A) Proximity ligation assay (PLA): HEK293T cells were treated as follows: vehicle – untreated cells; X/XO - cells treated with 8 μM xanthine plus 1 mU/mL xanthine oxidase for 24 h; auranofin - 0.8 μM auranofin for 24 h. The primary antibodies of Prdx2 and CSN5 were used at a 1:350 dilution. The presented images are representative of three biological independent experiments conducted in HEK293T wt cells. The PLA foci are shown in magenta, with some emphasised by red arrows. The DAPI-stained cellular nuclei are in blue. Scale bar: 10 μm. (B) The bar graphs quantify the number of PLA foci per nucleus by ImageJ software above a threshold of 1,500, representing the mean ± SD of data obtained from three independent experiments with overall n = 332 to 379 cell nuclei in each condition, respectively; the significance was calculated using a One-Way ANOVA; ****p < 0.0001 to every other columns. (C) HEK293T cells were transfected with BirA*-Flag-Prdx2, while non-transfected wt cells served as a negative control, and treated with PBS vehicle, 0.8 μM auranofin or 8 μM xanthine plus 1 mU/mL xanthine oxidase, as well as 50 μM biotin for 24 h. Cells were harvested, and PBS vehicle treated cells were resuspended in DMEM with or without 100 μM H₂O₂ for 2 min. Subsequently, all cells were resuspended in PBS containing 50 mM NEM for 5 min before lysis. Cell lysates were immunoprecipitated (IP) with Anti-FLAG® M2 Magnetic Beads and analysed by non-reducing SDS-PAGE followed by a western blot using anti-Prd×2 antibody and then stripped to reblot using anti-CSN5 antibodies. Sample in lane 6 is the sample of lane 5 reduced with 50 mM DTT. No CSN5 co-immunoprecipitated with BirA*-Flag-Prdx2. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)