Recombinant active full-length human Akt1/PKB protein (400 ng) (Cat#14–276, Millipore Sigma) was incubated with 200 μM ATP (Cat#20–306, Sigma) and 500 ng recombinant active full-length human AURKB protein (Cat#14–835, Millipore Sigma) in 50 μl 1 × kinase buffer (25 mM Tris-HCl (pH 7.5), 5 mM beta-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2, Cat#9802, Cell Signaling Technology). Reactions were incubated at 30°C for 30 min and terminated by addition of Laemmli SDS sample dilution buffer. Proteins were separated by 4–12% NuPAGE Bis-Tris gels, and phosphorylation of AURKB was visualized by immunoblotting with anti-Phospho-AKT Substrate (RXXS*/T*) (110B7E) Rabbit mAb (Cat#9614, Cell Signaling Technology).
Anti phospho akt substrate
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-Akt substrate is a laboratory reagent used to detect and analyze the phosphorylation of Akt substrate proteins. It is a specific antibody that recognizes the phosphorylated form of the Akt substrate, enabling researchers to study Akt signaling pathways and their role in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag m2, by Merck Group (2 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Mgcl2, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Las 4000 imaging system, by Fujifilm (1 mentions)
Anti myc, by Cell Signaling Technology (1 mentions)
Anti β tubulin antibody, by Developmental Studies Hybridoma Bank (1 mentions)
Las 4000, by Cytiva (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc, by BioLegend (1 mentions)
Anti cleaved parp, by Cell Signaling Technology (1 mentions)
L lactate assay kit, by Abcam (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti bax, by Cell Signaling Technology (1 mentions)
Anti pkm2, by Cell Signaling Technology (1 mentions)
Anti phospho gsk3β, by Cell Signaling Technology (1 mentions)
5 protocols using anti phospho akt substrate
1
Kinase assay of AKT1 and AURKB
2
Protein Extraction and Western Blotting
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Larvae were lysed in a lysis buffer (20 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM EGTA, 150 mM NaCl, 20 mM NaF, 1% Triton X-100, 1 μg/ml leupeptin, and 1mM PMSF) for 30–60 min on ice. After centrifugation for 15 min at 13,200 rpm, supernatants were reserved for SDS-PAGE analysis, and proteins were then transferred to nitrocellulose membranes (GE Healthcare, #BA85). Membranes were incubated in a blocking solution (Tris-buffered saline (TBS) containing 0.1% Tween-20, 5% BSA) for 1hr. The primary antibodies used were anti-LKB1 [22 (link)], anti-phospho-Thr196 SIK3 [39 (link)], anti-phospho-Ser239 HDAC4 (Cell Signaling Technology, #3443), anti-phospho-Akt substrate (Cell Signaling Technology, #9614), anti-FLAG-M2 (Sigma, #F1804), anti-Myc (Cell Signaling Technology, #2272), anti-AKH (a gift from Dr. Veenstra), and anti-β-tubulin antibody (Developmental Studies Hybridoma Bank, E7). Protein detection was done using the LAS-4000 imaging system (Fujifilm), and densitometric analysis was performed using Multi Gauge 3.0 software.
3
Limonin Regulates Glycolysis and Apoptosis in Liver Cancer
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Hep3B and HepG2 cells were obtained from the Cell bank of Chinese Academy of Sciences (Shanghai, People’s Republic of China). By following the instructions of supplier, Hep3B and HepG2 cells were cultured with RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) in a 37°C incubator with 5% CO2. Limonin was purchased from Selleck (Shanghai, People’s Republic of China). The primary antibodies used in Western blotting including anti-Glut1, anti-voltage-dependent anion channel 1 (VDAC-1), anti-LDHA, anti-PKM2, anti-cleaved caspase-3, anti-phospho-Akt substrate, anti-cleaved PARP, anti-Bax, anti-Bak, anti-cyto C, anti-Bcl-2, anti-Bcl-xl, anti-Akt, anti-phospho-Akt, anti-GSK3β, anti-phospho-GSK3β and the secondary HRP-conjugated goat anti-rabbit IgG were products of Cell Signaling Technology (Beverly, MA, USA). The anti-hexokinase-2 antibody, Glucose Uptake Assay and l-Lactate Assay Kits were products of Abcam (Cambridge, UK). The Annexin V-FITC was product of BioLegend (San Diego, CA, USA). For cell transfection, Myr-Akt1 was obtained from Addgene (Cambridge, MA, USA), and the Lipofectamine 3000 was a product of Thermo Fisher Scientific (Waltham, MA, USA).
4
Immunoprecipitation and Western Blotting Protocols
5
Immunoblot Analysis of Phosphorylated Proteins
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Crude cell lysates were prepared using trichloroacetic acid (TCA) as described previously (Tatebe and Shiozaki, 2003 (link)). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with primary antibodies diluted as follows; anti-phospho-p70 S6K (1:5000; cat. no. 9206, Cell Signaling Technology) for phospho-Psk1 (Thr-415) detection, anti-phospho-Akt substrate (1:4000; cat. no. 9614, Cell Signaling Technology) for phosphorylated Rps6 detection (Nakashima et al., 2010 (link)), anti-Psk1 (1:5000; Chia et al., 2017 (link)), anti-Spc1 (1:10 000; Tatebe and Shiozaki, 2003 (link)), anti-FLAG (1:5000; M2, Sigma-Aldrich), anti-myc (1:5000; 9E10, Covance), anti-phospho-4EBP1 (T37/46) (1:2500; cat. no. 2855, Cell Signaling Technology), anti-4EBP1 (1:2500; cat. no. 9452, Cell Signaling Technology), anti-GFP (1:2500; cat. no. 04404, Nacalai Tesque). Anti-rabbit IgG (H+L) HRP-conjugated (1:10 000; cat. no. W4011, Promega), anti-mouse IgG (H+L) HRP-conjugated (1:10 000; cat. no. W4021, Promega) and anti-rat IgG (H+L) HRP-conjugated (1:10 000; cat. no. 112-035-003, Jackson ImmunoResearch) were used as secondary antibodies. For the detection of the phosphorylated form of Maf1, proteins were separated by Phos-tag SDS-PAGE using an 8% SDS polyacrylamide gel containing 50 µM Phos-tag and 100 µM MnCl2.
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