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Phospho 4e bp1 s65

Manufactured by Cell Signaling Technology
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Phospho-4E-BP1 S65 is a lab equipment product from Cell Signaling Technology. It detects the phosphorylation of 4E-BP1 at serine 65.

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Lab products found in correlation

4e bp1, by Cell Signaling Technology (2 mentions) Total 4e bp1, by Cell Signaling Technology (2 mentions) Phospho crebs133, by Cell Signaling Technology (1 mentions) Phospho akt s473, by Cell Signaling Technology (1 mentions) γh2ax, by Cell Signaling Technology (1 mentions) Lamin b1, by Cell Signaling Technology (1 mentions) β tubulin, by Proteintech (1 mentions) Cyclin b1, by Santa Cruz Biotechnology (1 mentions) Phospho chk1 s345, by Cell Signaling Technology (1 mentions) Cellytic mt reagent, by Merck Group (1 mentions) Phospho atg14 s29, by Cell Signaling Technology (1 mentions) Phospho wee1 s642, by Cell Signaling Technology (1 mentions) Phospho atr s428, by Cell Signaling Technology (1 mentions) Phospho s6, by Cell Signaling Technology (1 mentions) Lc3a b, by Cell Signaling Technology (1 mentions) β actin, by Proteintech (1 mentions) Gapdh, by Proteintech (1 mentions) Flag tag (flag), by Merck Group (1 mentions) Eif4e, by Santa Cruz Biotechnology (1 mentions) Ha tag, by BioLegend (1 mentions)

Close spelling variants of this product

4E-BP1 (409 citations) Anti-phospho-S65 4E-BP1 (2 citations)

4 protocols using phospho 4e bp1 s65

1

Comprehensive Western Blot Analysis

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Cells were lysed on ice with CelLytic MT reagent (Sigma) supplemented with protease and phosphatase inhibitors. Primary antibodies: TSC1 (#6935), TSC2 (#4308), phospho-S6 (#4858), S6 (#2217), p21 (#2947), Lamin B1 (#12586), LC3 A/B (#12741), phospho-ATG14-S29 (#92340), ATG14 (#96752), phospho-CREB-S133 (#9198), CREB (#9197), phospho-CHK1-S345 (#2348), phospho-WEE1-S642 (#4910), WEE1(#13084), PLK1 (#4513), γH2A.X (#9718), phospho-S6K-S371 (#9208), S6K (#2708), phospho-4EBP1-S65 (#9451), 4EBP1 (#9644), phospho-AKT-S473 (#4060), AKT (#4691), p53 (#2527), phospho-CDC2-T15 (#9111), CDC2 (#9116), and phospho-ATR-S428 (#2853) from Cell signaling (all used at a 1: 1000 dilution); β-tubulin (#10094-1-ap, 1: 4000), β-actin (#20536-1-ap, 1: 4000), and GAPDH (#10494-1-ap, 1: 10000) from ProteinTech; ATR (#A300-137A-T, 1: 1000) from Bethyl Lab, and CHK1 (#sc-8408, 1:1000) and Cyclin B1(#sc-245, 1: 1000) from Santa Cruz.
Liu Y., Azizian N.G., Sullivan D.K, & Li Y. (2022). mTOR inhibition attenuates chemosensitivity through the induction of chemotherapy resistant persisters. Nature Communications, 13, 7047.
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2

eIF4E and 4E-BP1 Expression Plasmids

Cited 1 time
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HA-tagged and FLAG-tagged eIF4E expression plasmids were constructed by cloning eIF4E to AfeI and SbfI sites on the pLVX-EF-puro plasmid (53 (link)). pLVX-EF-4E-BP1WT and pLVX-EF-4E-BP1S83A expression plasmids were generated based on previous constructs (34 (link)). Doxycycline-inducible 4E-BP1T37A and 4E-BP1T46A plasmids were constructed by cloning corresponding 4E-BP1 mutant fragments to AfeI and SbfI sites on the pLenti-TRE-MCS-EF-Puro-2A-rTet plasmid (54 (link)). DNA constructs used in this study are listed in Table S1.
The following primary antibodies were used in this study: total 4E-BP1 (53H11, Cell Signaling Technology), phospho-4E-BP1T37/T46 (236B4, Cell Signaling Technology), phospho-4E-BP1T70 (9455, Cell Signaling Technology), phospho-4E-BP1S65 (9451, Cell Signaling Technology), phospho-4E-BP1S83 (ABE2889, Millipore), eIF4E (C46H6, Cell Signaling Technology), eIF4GI (D6A6, Cell Signaling Technology), eIF4E (A-10, Santa Cruz Biotechnology), eEF2 (2332, Cell Signaling Technology), HA tag (16B12, BioLegend), and FLAG tag (M2, Sigma-Aldrich).
Sun R., Cheng E., Velásquez C., Chang Y, & Moore P.S. (2019). Mitosis-related phosphorylation of the eukaryotic translation suppressor 4E-BP1 and its interaction with eukaryotic translation initiation factor 4E (eIF4E). The Journal of Biological Chemistry, 294(31), 11840-11852.
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3

Western Blot Analysis of Cellular Proteins

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Cells were lysed in cell lysis buffer (20 mM Tris, pH 7.5, 135 mM NaCl, 5% [v/v] glycerol, 50 mM NaF, 0.1% [v/v] Triton X-100, plus protease inhibitors), centrifuged and protein quantified using Bradford reagent (Sigma-Aldrich). Samples were made up in NuPAGE LDS sample buffer (Life Technologies). Western blotting was performed as previously described [28 (link)]. Blots shown are representative of 3 independent experiments. Anti-β-actin (#4967), phospho-TSC2 S1387 (#5584), total TSC2 (#3990), IRE1α (3294S), phospho-S6K1 T389 (#9205), total S6K1 (#9202), phospho-rpS6 S235/236 (#2211), total rpS6 (#2217), phospho-4E-BP1 S65 (#9451), total 4E-BP1 (#9644), phospho-ACC S79 (#3661), total ACC (#3676), PARP (#9542), caspase 3 (#9662), ATF4 (#11815), phospho-FOXO3a (#9466S), total FOXO3a (#9467), phospho-PRAS40 T246 (#2997), total PRAS40 (#2691) and phospho-Bad S136 (#4366) antibodies were from Cell Signaling Technology (Danvers, MA, USA). GADD34 antibody (10449-1-AP) was from Proteintech (Manchester, UK).
Dunlop E.A., Johnson C.E., Wiltshire M., Errington R.J, & Tee A.R. (2017). Targeting protein homeostasis with nelfinavir/salinomycin dual therapy effectively induces death of mTORC1 hyperactive cells. Oncotarget, 8(30), 48711-48724.
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4

Immunoblotting Analysis of Lysosomal and Cellular Signaling

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Immunoblotting was performed on whole-cell lysates, lysosomal extracts as previously described (11 (link)). Cell Signaling Technology antibodies: Rheb (#13879), β-actin (#3700), 4E-BP1 (#9644), phospho-4E-BP1 S65 (#13443), S6K (#2708), phospho-S6K T389 (#9206), S6 (#2317), phospho-S6 S240_S244 (#5364), p18 (#8975), RagA (#4357), RagC (#3360), mTOR (#2983), ATG5 (#2630), Cathepsin D (#2284), Caspase-7 (#12827) and phospho-H2AX (#9718). Galectin-3. LAMP2 was purchased from Santa Cruz. LC3B antibody was generated as described previously (48 ). Lysosomal and extra-lysosomal fractions were purified according to the manufacturer’s instructions (Sigma #LYSISO1).
Rebecca V.W., Nicastri M.C., McLaughlin N., Fennelly C., McAfee Q., Ronghe A., Nofal M., Lim C.Y., Witze E., Chude C.I., Zhang G., Alicea G.M., Piao S., Murugan S., Ojha R., Levi S.M., Wei Z., Barber-Rotenberg J.S., Murphy M.E., Mills G.B., Lu Y., Rabinowitz J., Marmorstein R., Liu Q., Liu S., Xu X., Herlyn M., Zoncu R., Brady D.C., Speicher D.W., Winkler J.D, & Amaravadi R.K. (2017). A unified approach to targeting the lysosome’s degradative and growth signaling roles. Cancer discovery, 7(11), 1266-1283.
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