Fig 1: Identification of plasma membrane calcium ATPase isoform PMCA4b as a renalase binding protein.A, HK-2 cells incubated with either labeled RP-Scr220 or RP-220, biotin-labeled proteins purified using streptavidin column, separated by SDS-PAGE and visualized by western blot using streptavidin-HRP; * = regions evaluated by mass spectrometry in samples labeled with either RP-Scr220 or RP-220; # = RP-220 band containing the plasma membrane calcium ATPase isoform PMCA4b. B, Endogenous expression of PMCA4b in HK-2 cells, western immunoblot using isoform specific monoclonal; CCL-119: human leukemic cell line; thyroid tumor = human thyroid tumor cell line (ATCC, CRL-1803) 10 μg protein loaded in each lane. C, co-immunolocalization of PMCA4b and renalase in HK-2 cells, images acquired using a Zeiss laser scanning confocal microscope, scale bar = 9 μm; arrow = plasma membrane. D, Co-Immunoprecipitation of PMCA4b and renalase from HK-2 cell lysates; renalase-Ab-beads = renalase antibody coated beads; PMCA4b-Ab-beads = PMCA4b antibody coated beads.