Anti-DNase II polyclonal antibody detects recombinant and endogenous DNase II proteins.

A, Mock transfected (lane 1, 3) or transiently transfected (lane 2, 4) 293FT cells with a plasmid for expression of the mouse DNase II protein with a FLAG-His tag at the carboxyl-terminus. The cell lysates were analyzed by Western blotting using anti-FLAG M2 antibody (lanes 1, 2) or anti-DNase II antibody (lanes 3, 4). Closed arrowhead; DNase II-FLAG-His protein detected by both antibodies, open arrowheads; DNase II-FLAG-His protein detected only by anti-DNase II antibody. Asterisk indicates non-specific detection of protein bands. B, Analyses of Con A-eluted fractions. Spleen lysates from DNase II^+/+ IFN-IR^−/− mice and DNase II^−/− IFN-IR^−/− mice were partially purified with Con A Sepharose. Fractions eluted with 0.1 (lanes 1, 6), 0.2 (lanes 2, 7), 0.3 (lanes 3, 8), 0.4 (lanes 4, 9), and 0.5 M (lanes 5, 10) α-methyl-D-mannoside were concentrated using a trichloroacetic acid precipitation, and the samples were analyzed by Western blotting using either the anti-DNase II antibody (upper panel), or anti-cathepsin D antibody (lower panel). Cathepsin D was used to confirm that a lysosomal glycosylated protein was extracted in the lysates and purified by Con A Sepharose. C, DNase activity of the eluted fractions. The eluted fractions from Con A Sepharose were directly assayed for DNase activity as described in the experimental procedures. N: negative control experiment without lysates.