Alisertib was prepared and stored according to the manufacturer’s instructions (Millennium Pharmaceuticals, Inc., Cambridge, MA). Specific antibodies against p-MTOR(Ser2448), p-MEK1/2(Ser217/221), p-AKT(Ser473), p-AURKA(T288), mTOR, MEK, AKT, AURKA, RPS6KB1, p-RPS6KB1(T389), Phospho-(Ser/Thr) Phe, and β-Actin were purchased from Cell Signaling Technology (Beverly, MA). Recombinant human AURKA and RPS6KB1 proteins were obtained from Cell Sciences (Canton, MA). Specific antibodies against KRAS were purchased from Santa Cruz Biotechnology (Dallas, TX). KRAS-G12D lentiviral vector was purchased from Applied Biological Materials (Richmond, BC, Canada). Tet-One™ Inducible Expression System and Tet System Approved FBS were purchased from Clontech (Palo Alto, CA). Transfection reagent LipoJet was purchased from SignaGen Laboratories (Gaithersburg, MD). Tet-on expression system, doxycycline-free fetal bovine serum, and human cell cycle arrays were purchased from Clontech (Palo Alto, CA).
Phospho ser thr phe
Manufactured by Cell Signaling Technology
Phospho-(Ser/Thr) Phe is an antibody that recognizes proteins phosphorylated on serine or threonine residues followed by a phenylalanine residue. It is used for the detection and analysis of phosphorylated proteins in cell and tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Tetone inducible expression system, by Takara Bio (1 mentions)
P rps6kb1 t389, by Cell Signaling Technology (1 mentions)
Lipojet, by SignaGen (1 mentions)
Tet system approved fbs, by Takara Bio (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Aurka, by Cell Signaling Technology (1 mentions)
Rps6kb1, by Cell Signaling Technology (1 mentions)
Erastin s7242, by Selleck Chemicals (1 mentions)
Acetylated lysine, by Cell Signaling Technology (1 mentions)
Ki67 sc15402, by Santa Cruz Biotechnology (1 mentions)
Fer 1 s7243, by Selleck Chemicals (1 mentions)
Phosstop, by Roche (1 mentions)
Ndufs3, by Abcam (1 mentions)
Proteinase inhibitor cocktail, by Roche (1 mentions)
Hsc70 b6, by Santa Cruz Biotechnology (1 mentions)
Ndufv1, by Proteintech (1 mentions)
Phospho p38 mapk, by Cell Signaling Technology (1 mentions)
3 protocols using phospho ser thr phe
1
Alisertib-Mediated Signaling Pathway Study
2
Comprehensive Reagents for Cell Signaling
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The antibodies to DJ-1 (#5933), p-p38 (#9216), p38 (#8690), AHCYL1 (#94248), SLC7A11 (#12691), Acetylated-Lysine (#9441), and Phospho-(Ser/Thr) Phe (#9631) were obtained from Cell Signaling Technology. The primary antibody against NRF2 (sc13032), γ-GCS (sc390811), GSS (sc166882), SAHH (sc271389), and Ki67 (sc15402) were obtained from Santa Cruz Biotechnology. The primary antibody against GAPDH (db106), HA (db2603), and Flag (db7002) were obtained from Diagnostic Biosystems. GSH (G4251), NAC (A7250), DL-Met (M9500), L-glutamine (G3126), SAM (A4377), DL-homocysteine (H4628), SAH (A9384), and BSO (19176) were obtained from Sigma-Aldrich. Erastin (S7242), Fer-1 (S7243), and RSL3 (S8155) were obtained from Selleck Chemicals. Sorafenib (S125098) was obtained from Aladdin. ML210 (GC18705) was obtained from Glpbio. PE (HY100887) was obtained from MedChem Express. DMEM (GIBICO, #12800), RPMI-1640 (GIBICO, #31800), and DMEM without Met (#21013024) were obtained from Thermo Fisher Scientific.
3
Worm Protein Extraction and Western Blotting
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Protein sample preparations were performed by collecting worms from three full plates and whasing them at least three times in M9 buffer. Worm pellet was frozen in liquid nitrogen and, depending on the amount of worm pellet, liquefied in 100–200 μM worm lysis buffer (25 mM Tris-HCL pH 7.4, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 0.5% SDS, 10 mM DTT and proteinase inhibitor cocktail (Roche)). PhosStop (Roche) was added when phospho-antibody was used. Samples were sonicated using the BioruptorR Sonication device (Diagenode) six times at 30/60 cycles. Samples were then centrifugaed for 15 min at 16,000×g at 4 °C and the supernatant was used. Protein concentration was measured with Bradford assay. Western blotting was performed using antibodies against ATP-1 (ATP5A, 1:2000 Abcam, #ab14748), GFP (1:2000, OriGene, #TP401), HSP-1 (HSC70 (B6), 1:4000, Santa Cruz, #sc-7298). LONP-1 (1:2000 Proteintech, #15440-1-AP), NUO-1 (NDUFV1, 1:2000 Proteintech, #11238-1-AP), NUO-2 (NDUFS3, 1:2000 MitoSciences, #MS112), Phospho-p38 MAPK (1:2000, Cell Signaling, #4511) and Phospho-(Ser/Thr) Phe (1:2000, Cell Signaling, #9631). Data were normalized to HSP-1.
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