Quantifying Protein Expression in Cell Culture

Protein isolation from cell culture experiments, SDS-PAGE, and Western Blot were described elsewhere^{35 (link)}. The method involves cell lysis with CellLytic M lysis buffer (Sigma-Aldrich), protein separation using ClearPAGE 10% sodium dodecylsulfate polyacrylamide gels (C.B.S. Scientific), and sample transfer to PVDF membranes (Fisher Scientific). For detection of PLA2R1, primary recombinant rabbit monoclonal antibody (AB; ab211573, Abcam, Cambridge, UK) and a secondary horseradish peroxidase conjugated goat anti-rabbit IgG (sc-2004, Santa Cruz) were used. Actin was detected using primary mouse monoclonal AB (MAB1501R, Merck Millipore, Darmstadt, Germany) and secondary horseradish peroxidase conjugated goat anti-mouse IgG (sc-2005, Santa Cruz). Protein isolation, SDS-PAGE and Western Blot were repeated three times using separate cell culture passages (biological n = 4).