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. 1998 Apr 28;95(9):5021–5026. doi: 10.1073/pnas.95.9.5021

Figure 4.

Figure 4

Immunological detection of Cak1At and assay for CDK2 kinase, CTD kinase, and CAK activities. (A) Twenty-five micrograms of Arabidopsis crude protein extract (lane 1) and 10 μg of total protein extract from S. cerevisiae GF2351 cells carrying pYX112-cak1At (lane 2) or the empty pYX112 vector (lane 3) was immunoblotted with the anti-Cak1At antibody. (B) (1) Immunoprecipitates of Arabidopsis proteins with preimmune serum (lane 1) or the anti-Cak1At antibody (lane 2) were subjected to immunoblotting with the anti-Cak1At antibody. (2) Immunoprecipitates of Arabidopsis proteins with preimmune serum or the anti-Cak1At antibody were assayed for CDK2 kinase activity with GST-CDK2 (K33R) (lane 1) or GST-CDK2 (T160A) (lane 2). (3) Immunoprecipitates of Arabidopsis proteins obtained with preimmune serum or the anti-Cak1At antibody were assayed for CDK2-activating kinase (CAK) activity, using GST-CDK2 (wild type) (lane 1), GST-CDK2 (K33R) (lane 2), or GST-CDK2 (T160A) (lane 3) as substrates. (C) (1) Ten micrograms of K1 and K2 fractions of Arabidopsis proteins was immunoblotted with the anti-Cak1At antibody. (2) K1 and K2 fractions of Arabidopsis proteins were mixed with GST-CTD, and CTD-associated proteins were subjected to phosphorylation reaction. (D) (1) Arabidopsis total protein (lane 1), p13suc1-associated proteins (lane 2), and the supernatant after depletion of p13suc1-associated proteins (lane 3) were immunoblotted with the anti-Cak1At antibody. (2) p13suc1-associated proteins were assayed for CDK2 or CTD kinase activity.