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. 1999 Nov;10(11):3863–3876. doi: 10.1091/mbc.10.11.3863

Figure 2.

Figure 2

Expression of endogenous PAP2b. (A) Tissues from a female adult mouse were dissected, washed in PBS, and Dounce-homogenized 10–20 strokes in TSE buffer (50 mM Tris, pH 7.4, 250 mM sucrose, and 1 mM EDTA) on ice. Homogenates were centrifuged at 2000 × g for 15 min to remove debris. One hundred micrograms of protein from the indicated tissue homogenate were separated (12.5% SDS-PAGE), transferred, and analyzed for PAP2b by Western blotting. (B) PAP2b was immunoprecipitated from 300 μg of protein from Swiss 3T3 cell detergent lysate and assayed for PAP activity using 100 μM PA/1 mM TX-100 per assay for 30 min at 37°C. “Mock” represents immunoprecipitation using an irrelevant IgG. (C) Each IP from B was separated (12.5% SDS-PAGE), transferred, and analyzed for PAP2b by Western blotting. Results are expressed as the means of triplicate immunoprecipitates (IP). Results are representative of three independent experiments.