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. 2011 Nov 18;11(2):M111.010132. doi: 10.1074/mcp.M111.010132

Fig. 3.

Fig. 3.

Effects of PKA and/or PKC on in vitro reconstituted UbcH5a/CHIP-mediated CYP3A ubiquitination. A, ubiquitination of CYP3A4 in an in vitro reconstituted UbcH5a/CHIP system PhosphorImager analyses of UbcH5a/CHIP-mediated ubiquitination of CuOOH-inactivated CYP3A4 in the presence or absence of PKA and/or PKC or in the presence or absence of ATP in the incubation. CYP3A4 was immunoprecipitated from the incubation mixture at 90 min of incubation and subjected to SDS-PAGE analyses. For details of the complete CYP3A4 ubiquitination analyses, see “Experimental Procedures.” The data from a typical experiment are depicted. Lane 1 corresponds to the complete reaction mixture with ATP, but no CYP3A4, PKA, or PKC. Lanes 2–5 correspond to the complete ATP-supplemented CYP3A4 ubiquitination system in the presence of no kinase (2), either PKA (3) or PKC (4) or both kinases (5). Lane 6 corresponds to the complete reaction mixture with CYP3A4 and PKA/PKC, but no ATP. Each experiment was conducted in its entirety at the least three separate times. B, ubiquitination of CYP3A4 or mutants in an in vitro reconstituted UbcH5a/CHIP system. Analyses of CYP3A4WT or its Ser-478, Thr-264, and Ser-420 mutants incubated for 90 min in a functionally reconstituted UbcH5a/CHIP ubiquitination system similar to that in A, and CYP3A4 immunoprecipitates were directly subjected to SDS-PAGE and Typhoon scanning analyses. Data from a typical experiment are depicted. Lane 1 corresponds to the complete reaction mixture with ATP, but no CYP3A4, PKA, or PKC. Lanes 2–4 correspond to the complete ATP-supplemented ubiquitination system containing CYP3A4 or one its Ser/Thr mutants in the absence of both PKA and PKC. Lanes 5–9 are the corresponding incubations carried out in the presence of both kinases. Lane 10 corresponds to the complete reaction mixture with CYP3A4 and PKA/PKC, but no ATP. Each experiment was conducted in its entirety at least three separate times. The color wheel intensity code is as follows: white > magenta > red > orange > yellow > green > light blue > dark blue > black.