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FL-BLyS was eluted with 0.1 M glycine HCl pH 3.0 and dialyzed against PBS, incubated with polymyxin B-agarose beads (Sigma-Aldrich, 1 ml of 50% suspension per 15 mls) for I hour, sterile filtered and quantitated by spectrophotometry (1 OD280 = 1.15 mg/ml).
About 50% of the injected dose accumulates in the cortex within I hour of injection and remains in the kidneys for 24 hours.
Extracts were removed and extractions were repeated with additional ethanol:acetone overnight at 26°C and then with acetone alone for I hour at 26°C.
Single endpoint PCR was carried out on polyA mRNA for gene detection and relative quantitative comparison between baseline (0 time) and I hour activation (with PMA +I).
Gels were fixed with 0.1% hexadecyltrimethylammonium bromide for 3 hours, air-dried I hour, stained with 0.1% toluidine blue, 0.1% alcian blue in acetic acid:ethanol:water (0.1 5 5) for 30 minutes and destained for 30 minutes.
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