P 4e bp1 thr37 46

Manufactured by Cell Signaling Technology

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P-4E-BP1 (Thr37/46) is an antibody that detects the phosphorylation of eIF4E-binding protein 1 (4E-BP1) at Thr37 and Thr46 residues. 4E-BP1 is a regulator of protein synthesis and its phosphorylation leads to the release of the eIF4E translation initiation factor, thereby promoting protein translation.

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43 protocols using p 4e bp1 thr37 46

Immunoblotting and Immunohistochemistry Protocols

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The following primary antibodies were used in this study for immunoblotting: pRB^ser780 (CST-3590), pRB^ser807 (CST-8516), total-RB (CST-9309), cyclin D1 (CST-2922), cyclin D3 (CST-2936), pAKT^ser473 (CST-9271), pAKT^Thr308 (CST-9275), total-AKT (CST-9272), pEGFR^Tyr1068 (CST-3777), total-EGFR (CST-2232), pERBB2^Tyr1248 (CST-2243), total-ERBB2 (CST-4290), pERBB3^Tyr1222 (CST-4784), pIGF1R^Tyr1135 (CST-3918), pS6K^Ser235/236 (CST-2211), total-S6K (CST-2217), Raptor (CST-2280), RheB (CST-13879), p4EBP1^Thr37/46 (CST-2855), 4EBP1 (CST-9452), pSIN1^Thr86 (CST-14716), SIN1 (CST-12860), pER^ser167 (CST-5587), Rictor (CST-2114) and Deptor (SCT-11816) were purchased from Cell Signaling Technology. p107 (sc-318), p130 (sc-317), total-ER (sc-8002, F-10), ERBB3 (sc-415) and IGF1R (sc-713) were purchased from Santa Cruz Biotechnology. β-tubulin (T-9026) were from Sigma-Aldrich and Ki67 from Clinisciences. The following antibodies were used for immunohistochemistry: pERK1/2^Thr202/4 (CST-4370), pAKT^ser473 (CST-4060), pS6K^Ser235/6 (CST-4858), pmTOR^Ser2448 (CST-2976) and p4EBP1^Thr37/46 (CST-2855) were purchased from Cell Signaling Technology. Ki67 was purchased from Clinisciences. Reagents were obtained from the following sources: 17-β-oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) from Sigma-Aldrich, fulvestrant from Tocris, and neratinib and vistusertib from SelleckChem.

Pancholi S., Leal M.F., Ribas R., Simigdala N., Schuster E., Chateau-Joubert S., Zabaglo L., Hills M., Dodson A., Gao Q., Johnston S.R., Dowsett M., Cosulich S.C., Maragoni E, & Martin L.A. (2019). Combination of mTORC1/2 inhibitor vistusertib plus fulvestrant in vitro and in vivo targets oestrogen receptor-positive endocrine-resistant breast cancer. Breast Cancer Research : BCR, 21, 135.

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Western Blot Analysis of Glucose Signaling

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Western blot analysis was performed as described from skeletal muscle of mice that underwent an oral glucose tolerance test [26] (link). Ponceau staining was used to confirm equal protein loading [29] (link). The following antibodies used for immunoblot analysis were purchased from Cell Signaling Technology (Beverly, MA): Akt (#9272), p-Akt Thr³⁰⁸ (#4056), p-Akt Ser⁴⁷³ (#9271), GSK3β (#9315), p-GSK3β Ser⁹ (#9323), GS (#3839), p-GS Ser⁶⁴¹ (#3891), mTOR (#2983), p-mTOR Ser²⁴⁴⁸ (#5536), 4EBP1 (#9644), p-4EBP1 Thr^37/46 (#2855), p-p70S6K Thr³⁸⁹ and Thr⁴²¹/Ser⁴²⁴ (#2708), p70S6K (#9205), p-STAT1 Tyr⁷⁰¹ (#9171), STAT1 (#9172). The following antibodies were purchased from Abcam (Cambridge, UK): total OXPHOS Rodent WB Antibody Cocktail (ab110413), FOXO1 (ab12161), and FOXO3 (ab47409). Antibodies against GLUT4 (#07-1404, Millipore, Darmstadt, Germany) and Hexokinase 2 (kindly provided by Oluf Pedersen, University of Copenhagen) were used. Appropriate secondary mouse or rabbit antibodies were purchased from Bio-Rad. The immunoreactive proteins were quantified densitometrically utilizing Quantity One Software (Bio-Rad).

Lundell L.S., Massart J., Altıntaş A., Krook A, & Zierath J.R. (2018). Regulation of glucose uptake and inflammation markers by FOXO1 and FOXO3 in skeletal muscle. Molecular Metabolism, 20, 79-88.

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Immunoblotting Analysis of Apoptosis Signaling

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Protein extraction, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting were performed as described previously [50 (link)]. The primary antibodies used were as follows: PDK1, p-PDK1 (Ser241), PTEN, mTOR, p-mTOR (Ser2448), p-mTOR (Ser2481), AKT, p-AKT (Ser473), p-AKT (Thr308), P70S6K, p-P70S6K (Thr389), 4E-BP1, p-4E-BP1 (Thr37/46), PI3Kp110α, poly(adenosine diphosphate-ribose) polymerase (PARP) and Caspase -8, -9, and -3, were purchased from Cell Signaling Technology (Beverly, MA,USA). Monoclonal anti β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Yang C., Huang X., Liu H., Xiao F., Wei J., You L, & Qian W. (2017). PDK1 inhibitor GSK2334470 exerts antitumor activity in multiple myeloma and forms a novel multitargeted combination with dual mTORC1/C2 inhibitor PP242. Oncotarget, 8(24), 39185-39197.

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Antibody Validation for Cellular Studies

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Antibody to dGAPDH is kindly provided by Dr. Wanzhong Ge. Antibodies to Ace‐lys (9441), TFEB (4240), Ace‐H3 (K9) (9649), Ace‐H3 (K27) (4353), histone H3 (4499), 4E‐BP1 (9644), and P‐4E‐BP1 (Thr37/46) (2855) were purchased from Cell Signaling Technology; antibodies to IFT20 (13615), PDLIM1 (11674), β‐Actin (66009), and p62 (18420) were purchased from Proteintech; antibodies to GFP (M048‐3), α‐Tubulin (M175‐3), Myc (M192‐3S), Flag (M185‐3B), HA (M180‐3S), and GST (M071) were purchased from MBL; antibodies to LC3 (L7543) and Tau (T9450) were purchased from Sigma; antibodies to CTSD (136282), LAMP1 (17768), and GCN5 (365321) were purchased from Santa Cruz; antibody to EGFR (db1025) was purchased from Diagbio.

Wang Y., Huang Y., Liu J., Zhang J., Xu M., You Z., Peng C., Gong Z, & Liu W. (2019). Acetyltransferase GCN5 regulates autophagy and lysosome biogenesis by targeting TFEB. EMBO Reports, 21(1), e48335.

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Protein Expression Analysis in Hippocampal Slices

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Hippocampal slices were flash-frozen on dry ice and sonicated as previously described (Ma et al., 2013 (link)). Samples containing equal amounts of protein lysate were loaded on 4 – 12% Tris-glycine SDS-PAGE gels for standard gel electrophoresis. Membranes were probed overnight at 4°C using primary antibodies for the following antibodies: Kv4.2 (1:5000; Alomone Labs); GluA1 (1:1000; Cell Signaling); p-mTOR (Ser2448) (1:1000; Cell Signaling); mTOR (1:1000; Cell Signaling); p-p70S6K (Thr389) (1:1000; Cell Signaling); p70S6K (1:1000; Cell Signaling); p-4EBP1(Thr37/46) (1:1000; Cell Signaling); 4EBP1 (1:1000; Cell Signaling); p-Akt (Ser473) (1:1000; Cell Signaling); Akt (1:1000; Cell Signaling); p-GSK3β (Ser9) (1:1000; Cell Signaling); GSK3β (1:1000; Cell Signaling); p-eIF2α (Ser51) (1:1000; Cell Signaling); eIF2α (1:1000; Cell Signaling); p-eEF2 (Thr56) (1:1000; Cell Signaling); eEF2 (1:1000; Cell Signaling); eEF1A (1:5000; Millipore); p-AMPKα (Thr172) (1:1000; Cell Signaling); AMPKα (1:1000; Cell Signaling); GAPDH (1:10,000; Cell Signaling). Protein bands were visualized using chemiluminescence (Clarity™ ECL; Biorad) and the Biorad ChemiDoc™ MP imaging system. Densitometric analysis was performed using ImageJ. Data were normalized to GAPDH (for total proteins analysis) or relevant total proteins (for phospho proteins analysis) unless otherwise specified.

Day S.M., Yang W., Ewin S., Zhou X, & Ma T. (2017). Glucagon-Like Peptide-1 Cleavage Product GLP-1 (9-36) Amide Enhances Hippocampal Long-term Synaptic Plasticity in Correlation with Suppression of Kv4.2 expression and eEF2 Phosphorylation. Hippocampus, 27(12), 1264-1274.

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Immunoblotting Protein Detection Protocol

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Proteins from whole-cell lysates were detected by immunoblotting. Antibodies were from Cell Signaling Technology Inc. (Danvers, MA); p-AKT^Ser473 (#4060), p-4EBP1 (Thr37/46) (#2855), GAPDH (#2118), β-actin (#4970), p-NF-κB-p65 (#3033), p27^Kip1 (#3686), and p-IκBα (#2859). p-IKKα (S176/S180) was from R&D Systems (Minneapolis, MN). Rabbit-polyclonal-HBZ serum was previously reported,^{23 (link)} and Tax antibody was kindly gifted by Cynthia Masison (NCI/NIH). Anti-c-MYC [Y69] was from Abcam (Cambridge, MA). BATF3 (3H1) was from Novus Biologicals (Centennial, CO).

Daenthanasanmak A., Bamford R.N., Yoshioka M., Yang S.M., Homan P., Karim B., Bryant B.R., Petrus M.N., Thomas C.J., Green P.L., Miljkovic M.D., Conlon K.C, & Waldmann T.A. (2022). Triple combination of BET plus PI3K and NF-κB inhibitors exhibit synergistic activity in adult T-cell leukemia/lymphoma. Blood Advances, 6(7), 2346-2360.

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Western Blotting of Vascular Smooth Muscle Cells

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Western blotting assay was conducted as previously described (17 (link)). Extractions of VSMCs and artery tissues were lysed with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai). Protein samples were segregated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a poly-vinylidene fluoride membrane (Millipore, Billerica, MA). Primary antibodies against VRK1, β-catenin, phospho (p)-mTOR^Ser2448, mTOR, p-S6^Ser235/236, S6, p-4EBP1^Thr37/46, 4EBP1, Caspase 3, Caspase 9 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA). Quantitative analysis of protein band was performed by using Image J software (Bethesda, MA).

Sun X., Zhao W., Wang Q., Zhao J., Yang D, & Yang Y. (2022). Inhibition of VRK1 suppresses proliferation and migration of vascular smooth muscle cells and intima hyperplasia after injury via mTORC1/β-catenin axis. BMB Reports, 55(5), 244-249.

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Comprehensive Western Blot Antibody Panel

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This assay was performed as described previously [14 ]. Primary antibodies used in this study are: ER (6F11) from Abcam, Cambridge, MA, USA; PR (sc-7208) and BCL2 (sc-509) from Santa Cruz Biotechnology, Santa Cruz, CA, USA; P-AKT-Thr308 (#2214) and P-AKT-Ser473 (#2118) from Epitomics, Burlingame, CA, USA; P-PRAS40-Thr246 (#2997), P-GSK3-Ser9 (#9323), P-ERK1/2-Thr202/Tyr204 (#9101), P-S6-Ser240/244 (#2211), P-4EBP1-Thr37/46 (#9459), P-mTOR-Ser2448 (#2971), AKT (#9272), ERK1/2 (#9102), GSK-3β (#9832), PTEN (#9559), β-actin (#4970), and c-PARP (#9541) from Cell Signaling Technology, Danvers, MA, USA. All our shown Western blotting images are from the same gel with the same exposure to allow for a complete comparison between lines and across treatments.

Fu X., Creighton C.J., Biswal N.C., Kumar V., Shea M., Herrera S., Contreras A., Gutierrez C., Wang T., Nanda S., Giuliano M., Morrison G., Nardone A., Karlin K.L., Westbrook T.F., Heiser L.M., Anur P., Spellman P., Guichard S.M., Smith P.D., Davies B.R., Klinowska T., Lee A.V., Mills G.B., Rimawi M.F., Hilsenbeck S.G., Gray J.W., Joshi A., Osborne C.K, & Schiff R. (2014). Overcoming endocrine resistance due to reduced PTEN levels in estrogen receptor-positive breast cancer by co-targeting mammalian target of rapamycin, protein kinase B, or mitogen-activated protein kinase kinase. Breast Cancer Research : BCR, 16, 430.

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Evaluating Tumor Angiogenesis in A549 Xenografts

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Tumor sections from FFPE A549 xenograft tumors were stained for p-4E-BP1 [Thr37/46 (#2855), dilution 1:5000; Cell Signaling]. Detection was performed using CSA II signal amplification system (Dako). Sections were scored by two pathologists (H.M.H. and S.M.W.) using the semiquantitative H-score that takes into consideration the staining intensity (0–3+) in conjunction with the percentage of viable tumor cells staining positively. H-score = (% at 0) * 0 + (% at 1+) * 1 + (% at 2+) * 2 + (% at 3+) * 3. Thus, this score produces a continuous variable that ranges from 0 to 300.
Tumor vessel density was analyzed in FFPE A549 xenograft tumors. Sections were stained for CD31 [Anti-CD31 (#ab28364), dilution 1:50; Abcam]. Detection was performed using the EnVisionTM+ system (Dako). Two pathologists (H.M.H. and S.M.W.) counted the number of CD31-positive vessels in 10 randomly clockwise selected fields (200× magnification) containing viable tumor. Vessel density was calculated as the sum of CD31-positive vessels in the 10 fields.

Groenendijk F.H., Mellema W.W., van der Burg E., Schut E., Hauptmann M., Horlings H.M., Willems S.M., van den Heuvel M.M., Jonkers J., Smit E.F, & Bernards R. (2014). Sorafenib synergizes with metformin in NSCLC through AMPK pathway activation. International Journal of Cancer. Journal International du Cancer, 136(6), 1434-1444.

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Quantitative Protein Expression Analysis

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Qualitative analysis of protein expression and phosphorylation status was performed via standard western blotting procedures, as described previously (Durgan et al., 2011b (link)). Briefly, 20–30 µg protein lysate was separated on polyacrylamide gels and transferred to PVDF membranes. Membranes were probed for the following targets: p-STAT5^Tyr694 (Cell Signaling, 9351L), p-ERK 1/2^{Thr202/Tyr204} (Cell Signaling 9101), p-mTOR^Ser−2448 (Cell Signaling, 2974), and p-4EBP1^Thr−37/46 (Cell Signaling, 9459). Rabbit HRP-conjugated secondary antibodies (Cell Signaling 7076) were used for chemiluminescent detection with Luminata Forte Western Blotting substrate (Millipore, WBLUF0100). Densitometry data were normalized to total STAT5 (Santa Cruz Biotechnology, sc-835) or amido black. Importantly, in order to minimize the contribution that position on the gel might have on outcomes, samples were randomized on gels; samples were re-ordered post-imaging, only for the sake of illustration of representative images (note, all bands for representative images for an individual experiment were from the same gel; see Supplementary Figures S4, S5).

Sonkar R., Berry R., Latimer M.N., Prabhu S.D., Young M.E, & Frank S.J. (2022). Augmented Cardiac Growth Hormone Signaling Contributes to Cardiomyopathy Following Genetic Disruption of the Cardiomyocyte Circadian Clock. Frontiers in Pharmacology, 13, 836725.

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