Fig. 4.

Immunoclearing of Cbr1 and its effect on Doxol-forming activity in mouse liver cytosol. (A) Immunoblot analyses of Cbr1 in mock-cleared (−) and Cbr1-cleared (+) cytosols from six wild-type (wt) mice. Cbr1 protein levels were determined by densitometry, and the % Cbr1 protein remaining after immunoclearing, relative to mock-cleared samples, is shown at bottom. Trx1 levels were determined in parallel as a control. (B) NADPH-dependent Doxol-forming activity in mock-cleared and Cbr1-cleared cytosols. Reaction conditions were 200 µM NADPH, 200 µM Dox, and 12-µg equivalents of cytosolic protein; incubations were for 1 hour at 37°C. Doxol levels were measured by LC-MS/MS. The % Doxol-forming activity remaining after immunoclearing, relative to mock-cleared samples, is shown at bottom. Error bars represent 1 S.D.; n = 3 technical replicates for all reactions; *P < 0.05 by Student’s t test. (C) Mock-cleared and Cbr1-cleared Doxol-forming activity for the six biologic replicates. The concentration of Doxol generated is reported in arbitrary units (AU), where the average level in mock-cleared samples is set to 1. rror bars represent 1 S.D.; *P < 0.005 by Student’s t test.