Enzymatic Activity Assay of Cell Surface TPO

The enzymatic activity of cell surface-expressed TPO in the studied cell lines was analyzed as we had previously described [31 (link)]. Briefly, cells were grown 48 h in a medium supplemented with 20 mM hemin (Sigma-Aldrich). After washing with PBS, cells were incubated with reaction mixture containing Amplex Ultra Red (Life Technologies) in the presence of superoxide dismutase (SOD), KI, and H₂O₂ (Sigma-Aldrich). Reaction was stopped at 1-minute intervals by addition of catalase and SOD. The fluorescence was measured in the Synergy H4 hybrid multi-mode microplate reader (BioTek, USA) [31 (link)]. The enzymatic activity of TPO expressed in tissues was determined using a luminol-based assay described by Jomaa and collaborators [25 (link), 32 (link)], with some modifications. Briefly, breast tissue lysates (200 μg) were immunoprecipitated with mAb A4 (6 μg) and protein A agarose (Merck Millipore, Darmstadt, Germany). 50 μl of resin-bound TPO in 0.1 M Tris-Cl (pH 8.6) was incubated with 150 μl of 1.3 M glycine-NaOH (pH 9.0), 1.3 mM EDTA in a 96-well plate for 5 minutes. The reaction was initiated by adding 20 μl of 400 μM luminol (Sigma-Aldrich) in 1 M glycine-NaOH (pH 9.0), 1 mM EDTA, followed by the supplementation with 5 μl of 80 mM H₂O₂. Luminescence intensities were measured in the Synergy 2 instrument (BioTek).