GDC-0349 was from Dr. Zhou at Hubei Cancer Hospital11 (link). Antibodies of phosphorylated (“p”)-Akt (Ser-473) (#9271), Akt (Thr-308) (#13038), Akt1 (#75692), p-S6K1 (#9234), S6K1 (9202), p-JNK1/2 (#9255), JNK1/2 (#9252), SphK1 (#12071), cleaved-caspase-3 (#9664), cleaved-caspase-9 (#20750), cleaved-poly (ADP-ribose) polymerase (PARP) (#5625), and β-tubulin (#15115) were purchased from Cell Signaling Tech (Beverly, MA). All cell culture reagents were obtained from Hyclone Co. (Suzhou, China). N-acetylcysteine (NAC), sphingosine-1-phosphate (S1P) and SP600125, rapamycin, perifosine, AZD-2014, puromycin, and polybrene were purchased from Sigma-Aldrich (St. Louis, Mo). Primers, sequences and all viral constructs were designed and provided by Shanghai Genechem (Shanghai, China) unless otherwise mentioned.
Akt thr 308
Manufactured by Cell Signaling Technology
Sourced in China
Akt (Thr-308) is a phospho-specific antibody that recognizes Akt protein phosphorylated at threonine 308. Akt, also known as protein kinase B (PKB), is a serine/threonine protein kinase that plays a key role in multiple cellular processes, including cell proliferation, survival, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Gsk3β ser9, by Cell Signaling Technology (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Polybrene, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Puromycin, by Merck Group (1 mentions)
Cleaved poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
Perifosine, by Merck Group (1 mentions)
Cleaved caspase 9, by Cell Signaling Technology (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
Jnk1 2, by Cell Signaling Technology (1 mentions)
Sphk1, by Cell Signaling Technology (1 mentions)
P s6k1, by Cell Signaling Technology (1 mentions)
Azd2014, by Merck Group (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Total s6k1, by Cell Signaling Technology (1 mentions)
App c terminal, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Total gsk3β, by Cell Signaling Technology (1 mentions)
Total irs 1, by Cell Signaling Technology (1 mentions)
Totalpi3k p85, by Cell Signaling Technology (1 mentions)
4 protocols using akt thr 308
1
Signaling Pathway Regulation in Cancer
2
Western Blot Antibody Validation Protocol
3
Western Blot Analysis of Cell Signaling
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Lysates were extracted from cultured cells using T-PER Protein Extraction Reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing PhosSTOP (Roche, Basel, Switzerland) and Complete Mini. Equal amounts of proteins were electrophoresed and transferred to nitrocellulose membranes or polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). After they were blocked in 5% non-fat milk, the membranes were incubated with the following primary antibodies: BZW2 (polyclonal; 1:500; #21001-1-AP; Proteintech, Wuhan, China), mTOR (total) (monoclonal; 1:1,000; #2983), mTOR (Ser2448) (monoclonal; 1:1,000; #5536), Akt (total) (monoclonal; 1:1,000; #4691), Akt (Thr308) (monoclonal; 1:1,000; #13038) (all from Cell Signaling Technology), or β-actin (polyclonal; 1:5,000; #20536-1-AP; Proteintech). The secondary antibody was goat anti-rabbit IgG (1:5,000; #A0545; Sigma-Aldrich). Visualization was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Inc.).
4
Proximity Ligation Assay for Protein Interactions
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Fixed cells were blocked (Olink) on-chip for 1 h. Next, monoclonal primary antibodies (Cell Signaling Technology) were diluted with antibody diluent (Olink) and incubated with the cell samples for 12 h with refresh cycles repeated every 2 h. The dilution ratio was 1 : 50 for Akt (Ser-473) (Cell Signaling, 4060), Akt (Thr-308) (Cell Signaling, 2965), and GSK-3β (Ser-9) (Cell Signaling, 9323), 1 : 25 for the PDGF β receptor (Tyr-751) (Cell Signaling, 3161), and 1 : 250 for the IGF-1 receptor (Tyr-1161) (Santa Cruz Biotechnology). Anti-rabbit PLA probes (anti-rabbit Plus and Minus, Olink) were diluted with Duolink antibody diluent at a ratio of 1 : 5 and incubated at room temperature for 2 h. DNA connectors (Olink) with the T4 ligase (Fermentas) were incubated at 32 °C for 1 h. Amplification reagents (Olink) were diluted according to the vendor's instructions and incubated at 32 °C for 2 h. Cell nuclei and cytoskeletons were counterstained with 4′,6-diamidino-2phenylindole dihydrochloride (DAPI) (Sigma Aldrich) and Phalloidin-Atto 488 (Sigma Aldrich), respectively.
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