Fig. 5.

Immunoclearing Cbr3 and Tr1 from mouse liver cytosols and effect on Dox-dependent NADPH oxidation and NADPH-dependent Doxol formation. (A) Immunoblot analysis of Cbr3 and Tr1 in mock-cleared (−) and immunocleared (+) mouse liver cytosols. The Cbr3 immunoblots were probed with either raw anti-Cbr3 antiserum (top) or Cbr3-specific antiserum (bottom). (B) NADPH-dependent Doxol formation by mock-cleared and Cbr3-cleared cytosols. Reaction conditions were 200 µM NADPH, 200 µM Dox, and 45-µg equivalents of cytosolic protein; incubations were for 1 hour at 37°C. Doxol levels were measured by LC-MS/MS. Triplicate incubations for each cytosol were pooled and analyzed as one sample. (C) Mock-cleared and Cbr3-cleared Doxol-forming activity for the three 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. Error bars represent 1 S.D.; there was no significant difference. (D) Dox-dependent NADPH oxidation by mock-cleared and Tr1-cleared cytosols. Reaction conditions were as described in (B). Error bars represent 1 S.D.; n = 3 technical replicates for all reactions. (E) Mock-cleared and Tr1-cleared Dox-dependent NADPH oxidation for the three biologic replicates. The change in NADPH is reported in arbitrary units (AU), where the average change in mock-cleared samples is set to 1. Error bars represent 1 S.D.; there was no significant difference.