Identification of 57-kDa PXDNL in different cell lines. (A) Thirty micrograms of cytoplasmic protein from MCF-7, MDA-MB-231, U2OS, HEK-293T, K562, MEL, and Cos-1 cells was separated on a 10% SDS-PAGE gel. A Western blot of this was probed first with an affinity-purified antibody to the peroxidase domain of human PXDNL (upper panel) and then stripped and probed with antibody to human GAPDH (lower panel) as a loading control. Note that the anti-human antibodies react poorly with both murine proteins (lane 6). (B) Cos-1 cells were transiently transfected with a plasmid expressing 57-kDa PXDNL with an N-terminal Flag tag and a C-terminal sequence that is biotinylated in vivo. Note that these two tags increase the size of the recombinant protein over endogenous 57-kDa PXDNL. Cytoplasmic extract was recovered with streptavidin paramagnetic beads (lane 2) or by immunoprecipitation with anti-PXDNL antibody (lane 3) or nonimmune IgG (lane 4), and a Western blot of input and bound proteins was probed with antibody to the Flag tag. The same relative amount of input and streptavidin-recovered protein (1/10th of total sample) and 100% of the antibody bound samples were applied to the gel.
