Anti-Cyclin-dependent kinase 1 CDK1 Antibody Picoband®

EV-D68 infection prevents cell exit from G0/G1 into S phase and promotes G2/M to G0/G1 transition . (A) RD cells were serum-starved for 24 h and then mock-infected (mock) or infected with EV-D68 Fermon strain (infected) at an MOI of 0.8. After 2 h of virus adsorption, the cells were treated with medium without FBS for 18 h, followed by medium containing 10% FBS. Top panel: The cell cycle profiles were determined by flow cytometry at 0, 18, 24, and 30 h post-infection. Bottom panel: The histograms show the percentage of cells in each phase of the cell cycle. (B) G0/G1 and S phase-related cell cycle proteins were analyzed by western blot analysis. RD cells were mock-infected (m) or infected with EV-D68 Fermon strain at an MOI of 0.8 (i) for 0, 16, 20, 24, and 28 h. Histone is shown as a loading control. Results are representative of three independent experiments. (C) At 24 h post-infection, mRNA levels of CDK2, cyclinE1, CDK4, CDK6, and cyclinD were evaluated in mock infected (mock) and EV-D68 infected (infected) cells by quantitative real-time PCR. The results are standardized to GAPDH and normalized to 1.0 in mock-infected cells. (D) RD cells were treated with 25 ng/mL nocodazole or control medium for 24 h, infected with EV-D68 Fermon strain at an MOI of 0.8 or mock for 2 h and then re-treated with 25 ng/mL nocodazole for synchronization. Top panel: Cell-cycle profiles were determined by flow cytometry. Low panel: The histograms show the percentage of cells in each phase of the cell cycle. (E) G2/M phase-related proteins were detected by western blot analysis. Results are representative of three independent experiments. (F) At 24 h post-infection, mRNA levels of CDK1 and cyclinB1 were assessed in mock-infected (mock) and EV-D68 infected (infected) cells by quantitative real-time PCR. The results are standardized to GAPDH mRNA and normalized to 1.0 in mock-infected cells. (G) At 28 h post-infection, the protein concentration of CDK4, CDK6, cyclinD1, CDK2, cyclinE1, CDK1, cyclinB1, and histone were assessed in mock-infected (mock) and EV-D68 infected (infected) cells by Elisa kit. The results are standardized to Histone (his). The results indicate the mean ± S.D of three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.
Index in PubMed under a CC BY license. PMID: 28229049

Western blot analysis of CDK1 using anti-CDK1 antibody (PA1544).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 30 ug of sample under reducing conditions.
Lane 1: human Hela whole cell lysates,
Lane 2: human K562 whole cell lysates,
Lane 3: human PC-3 whole cell lysates,
Lane 4: human SH-SY5Y whole cell lysates.
After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-CDK1 antigen affinity purified polyclonal antibody (Catalog # PA1544) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1002) with Tanon 5200 system. A specific band was detected for CDK1 at approximately 34 kDa. The expected band size for CDK1 is at 34 kDa.