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. 2021 Nov 25;10:e68473. doi: 10.7554/eLife.68473

Figure 1. Critical residues in CycT1 (Thr143 and Thr149) are required for its binding to CDK9.

Figure 1.

(A) Diagram of WT CycT1 and indicated mutant CycT1 proteins. The full-length human CycT1 protein contains 726 residues. Two cyclin boxes are found between positions 30 and 248. Critical residues for CDK9 binding include Thr143, Thr149, and Thr155. Glu137 and Leu203 flank these sites. Presented are critical mutations in CycT1 that form the basis of this study. (B) Mutant CycT1 proteins are unstable. WT CycT1 and three indicated mutant CycT1 proteins were expressed in 293T cells, which were untreated (lanes 1, 3, 5, and 7) or treated with 2 μM bortezomib for 12 hr (lanes 2, 4, 6, and 8) before cell lysis. Levels of CycT1 (panel 1), CDK9 (panel 2), and the loading control actin (panel 3) proteins were detected with anti-HA, anti-CDK9, and anti-β-actin antibodies, respectively, by WB. Top panels are designated as panel 1, and panel numbers increase from top to bottom (same numbering rules are applied to all WB panels throughout). Gels are marked as follows: IP, IPed proteins, above panels; next, presence and absence of co-IPed proteins is denoted by (+) and (-) signs; same for the inclusion and concentration of bortezomib; WB, western blot of co-IPed proteins; Input, western blot of input proteins. (C) Mutant CycT1 proteins are unstable. WT CycT1 and three indicated mutant CycT1 proteins were expressed in 293T cells, which were untreated (lanes 1, 5, 9, and 13) or treated with 100 μg/ml cycloheximide (CHX) for 3–9 hr (lanes 2–4; 6–8; 10–12; 14–16) before cell lysis. Levels of CycT1 (panel 1) and the loading control actin (panel 2) proteins were detected with anti-HA and anti-β-actin antibodies, respectively, by WB. (D) Interactions between CDK9 and mutant CycT1 proteins are impaired. WT CycT1 and three indicated mutant CycT1 proteins were expressed in 293T cells treated with bortezomib. Co-IPs with CDK9 are presented in panels 1 and 2. Panels 3 and 4 contain input levels of CycT1 and CDK9 proteins. (E) Interactions between CDK9 and point mutant CycT1 proteins are impaired. WT CycT1 and four indicated mutant CycT1 proteins were expressed in 293T cells treated with bortezomib. Co-IPs with CDK9 are presented in panels 1 and 2. Panels 3 and 4 contain input levels of CycT1 and CDK9 proteins. (F) Interactions between CDK9 and truncated mutant CycT1(280) proteins are impaired. WT CycT1 and three indicated mutant CycT1 proteins were expressed in 293T cells treated with bortezomib. Co-IPs with CDK9 are presented in panels 1 and 2. Panels 3 and 4 contain input levels of CycT1(280) and CDK9 proteins.