Mab2871

Nicotiana benthamiana ΔXTFT plants were grown in a growth chamber at 22°C with a 16-h light/8-h dark photoperiod. All fusion proteins and antibodies or antibody fragments were transiently expressed via agro-infiltration. Total soluble proteins (TSP) and proteins secreted to the apoplastic fluid (AF) were collected 4–5 days post-infiltration (Kriechbaum et al., 2020 (link)). Expression of EPO-fusions was analyzed by immunoblotting of SDS–PAGE or native PAGE using mouse anti-EPO (1:5000, MAB2871, R&D Systems, Minneapolis, MN, United States), anti-human IgG-HRP (HC and LC, 1:10,000, W4031, Promega, Mannheim, Germany), anti-mouse IgG-HRP (1:10,000, Sigma-Aldrich, A0545), and anti-human kappa-chain-HRP (LC, 1:1000, Sigma-Aldrich, A7164) antibodies. EPO-Fc variants were purified from agroinfiltrated leaves (800 mg) with rProtein A or Protein G Sepharose^TM Fast Flow (GE Healthcare) and with KappaSelect^TM (GE Healthcare) for EPO-CL, according to the manufacturer’s instructions. Heavy chains of cetuximab (Castilho et al., 2015 (link)), anti-ebola 13F6 (Castilho et al., 2011a (link)) antibodies, and 2G12-ScFvFc (Loos et al., 2011 (link)) were cloned previously, and their expression was analyzed with anti-human gamma-chain-HRP (HC, 1:1000, Sigma-Aldrich, A7164). All purified proteins were stained with Coomassie Brilliant Blue.

EPO^−/−, WT controls, and HEK-293 cells transfected with hEPO overexpression vector⁴⁹ (link) were subjected to immunoblot analysis, as described previously,⁶² (link) using a monoclonal mouse anti-EPO antibody (1:1,000; MAB2871, R&D systems, Abingdon, UK)⁶³ (link) and a mouse anti-GAPDH antibody (1:1,000; sc-47724, Santa Cruz Biotechnology, Texas, USA). Goat anti-mouse IgG (H + L) Cross-Absorbed Alexa Fluor 680 (1:5,000; A21057, Invitrogen, Massachusetts, USA), was used as the secondary antibody. Membranes were visualized on the LI-COR Odyssey CLx system (LI-COR Biotechnologies, Nebraska, USA). Images were converted to grayscale using Image Studio Lite version 5.2.5 (LI-COR Biotechnologies, Nebraska, USA).