Rabbit anti phospho ampk thr172

Hela cells grown on glass coverslips were treated by C5M (100 nM) or water (control, Co) in different trials and then, fixed by formaldehyde 4% for 10 minutes at 37°C. Immunofluorescences were performed as described previously [40 (link)]. Coverslips were incubated with the antibodies directed against the following antigens: phospho-histone H3-Serine10 (Upstate, 1:2000); phospho-histone H3-Serine 28 (Upstate, 1:2000); aurora kinase B (Epitomics, 1:2000); rabbit anti-phospho-AMPK (Thr172) (Cell Signaling, 1:100), rabbit anti-phospho-aurora A/B/C (Cell Signaling, 1:100). Actin was stained by phalloidin-rhodamin (1 μg/ml). DNA was visualized with Hoechst 33342 (Sigma, 0.5 μg/ml). Images were collected with a ZEISS 710 Laser Scanning Confocal microscope with a 63×-immersion oil objective. Slices of 0.5 μm are shown.
Metaphases spreading were realized as follows: HeLa cells were incubated ON with either C5M (200 nM) or taxol (33 nM) and were then recovered by flushing. An osmotic choc was realized (calf serum diluted in water 1:5) for 13 min, at 37°C, then the same volume of fixative (mixture of EtOH and acetic acid (3:1)) was added. After 20 min of incubation at room temperature, cells were washed by fixative and stored at 4°C. Finally cells were spread on cold coverglass and DNA was stained with Hoechst.

Cells treated by compounds or DMSO (control, Co) or PBS (Co) were harvested and lysed in 9M urea, finally supplemented with Laemmli sample buffer as described previously [40 (link)]. AMPK detections were performed on RIPA-cell extracts. Western blots were performed using the following antibodies: rabbit anti-phospho-histone H3 (Ser10) (Upstate, 1:2000); rabbit anti-aurora B (Epitomics, 1:5000); rabbit anti-phospho-AMPK (Thr172) (Cell Signaling, 1:1000); rabbit anti-AMPK (Cell Signaling, 1:1000); mouse Δ-actin (Sigma, 1:5000). Bands were visualized by horseradish peroxidase labelled antibodies and ECL technique (Amersham Bioscience). Images were observed by ChemiDoc MP system (Biorad).

Adult male C57BL/6N mice (body weight: 20–23 g, age: 6–7 weeks) were obtained from the Charles River (Beijing, China). Alexa Fluor 488‐ and 532‐labeled fluorescent secondary antibodies, BCA Protein Assay Kit, and other reagents were purchased from Thermo Fisher Scientific Inc (ThermoFisher Scientific, Waltham, MA, USA). The rabbit anti‐AMPK antibody (D5A2), rabbit anti‐SIRT1 antibody (D1D7), rabbit anti‐Phospho‐FoxO1 (Ser256), and rabbit anti‐Phospho‐AMPK (Thr172) were purchased from Cell Signaling Technology (Danvers, MA, USA). The RIPA lysate was purchased from Kangwei Century Biotechnology (Cwbio, Beijing, China). The Tunel assay kits (G1501) were purchased from Wuhan Servicebio Company (Wuhan, China). The MCAO thread plugs were purchased from RWD life science Co., Ltd. (Shenzhen, China). Compound C (866405–64‐3) was purchased from the MCE (Shanghai, China). Other chemical reagents were analytically pure.

Total protein content was extracted by RIPA lysis buffer containing Protease and Phosphatase Inhibitor Cocktail (MedChem Express, USA) and measured using a BCA Protein Assay Reagent kit (Beyotime Biotechnology, Shanghai, China). Protein expressions were detected by Simple Western using a Size Separation Master Kit with Split buffer (12–230 kD) following the instructions of the manufacturer. The reliability of this experimental approach has been verified by previous studies [33 (link),34 (link)]. In brief, 1.5 μg protein of each sample was separated by capillary electrophoresis on Wes instrument (Protein Simple, San Jose, CA, USA) under default settings. Protein levels were probed with rabbit anti-AMPK (1:50, #2532, Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-AMPK (Thr172) (1:50, #2535, Cell Signaling Technology, USA), and rabbit anti-β-actin (13E5) (1:50, #4970, Cell Signaling Technology, USA). An Anti-Rabbit Detection Module was purchased from Protein Simple. Data were analyzed by Compass software (Protein Simple, San Jose, CA, USA) version 5.0.1.