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. 2017 Jul 28;7:6796. doi: 10.1038/s41598-017-07179-8

Figure 3.

Figure 3

cdk9 interacts directly with CG16947. (a) Alignment of the Drosophila melanogaster (fly) and human protein sequences of cdk9. Highlighted portion denotes region that the commercial anti-cdk9 antibody was generated against human cdk9 protein. (b) CG16947 (50 kDa) was detected in the lysates from Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) and Repo-Gal4 > UAS-CG16947 (Repo > UAS-CG16947) used for co-immunoprecipitation in 3d, e, f and Supplemental Fig. 2. Kinesin (120 kDa) was used as a loading control to ensure that similar levels of protein were loaded from both genotypes. (c) cdk9 (49 kDa) was detected in the lysates from Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) and Repo-Gal4 > UAS-CG16947 (Repo > UAS-CG16947) used for co-immunoprecipitation in 3d, e, f and Supplemental Fig S2. Kinesin (120 kDa) was used as a loading control to ensure that similar levels of protein were loaded from both genotypes. (d) Western blot of anti-CG16947 co-immunoprecipitation. Flies were of either Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) or Repo > UAS-CG16947 (Repo > UAS-CG16947) genotype. Blot was probed with anti-cdk9. Lysate refers to the input for the co-immunoprecipitation. A control condition where the beads were incubated with the anti-CG16947 antibody in the absence of lysate was done to distinguish anti-CG16947 antibody elution from the beads versus the CG16947 protein itself. A 49 kDa cdk9 band was detected in the lysates and the without DTT elution of the anti-rchy1 pulldown in both Repo-Gal4 > UAS-GFP (Repo > UAS-GFP) and Repo-Gal4 > UAS-CG16947 (Repo > UAS-CG16947) conditions. (e) Co-immunoprecipitation with anti-cdk9. Anti-cdk9 pulled down CG16947 protein (50 kDa). An increase in the number and intensity of the bands recognised by anti-CDK9 in the MG132-treated samples compared to when the lysate and co-immunoprecipitation was done without MG132 suggest that CG16947 was actively degraded via the proteasome in vitro. The multiple bands observed below the expected size for CG16947 likely represent degraded CG16947, which can be observed when the proteasome is inhibited in vitro. (f) Co-immunoprecipitation with anti-cdk9. anti-cdk9 pulled down cdk9 protein that was ubiquitinated as shown by the bands above the expected size for cdk9, 49 kDa and degraded, as shown by the bands below 49 kDa. An increase in the number and intensity of the bands recognised by anti-ubiquitin in the MG132-treated samples demonstrated that cdk9 was actively ubiquitinated and degraded via the proteasome in vitro. A higher exposure of the blot is shown in Supplemental Fig. 2 where ubiquitinated cdk9 and its degraded products can be detected in the lysates without MG132.