Rabbit anti 4e bp1

Manufactured by Cell Signaling Technology

Sourced in United States, United Kingdom

Rabbit anti-4E-BP1 is a primary antibody that specifically recognizes the 4E-BP1 protein. 4E-BP1 is a regulator of protein translation initiation and is involved in the control of cell growth and proliferation.

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4E-BP1 (409 citations) Anti-4E-BP1 (139 citations) Rabbit anti-P-4E-BP1 (10 citations) Rabbit anti-p-4E-BP1 (Thr37/46) (4 citations) Rabbit anti-p-4E-BP1 (T37/46) (2 citations) Anti-4E-BP1 rabbit mAb (2 citations) Anti-rabbit phospho-4E-BP1 (Thr37/46) (2 citations)

38 protocols using rabbit anti 4e bp1

Western Blot Analysis of Protein Signaling

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Western blot analysis was performed as described previously (12 (link)) using the following antibodies: mouse anti-AKT (BD Biosciences), rabbit anti-pAKT, rabbit anti-p4E-BP1, rabbit anti-4E-BP1, rabbit anti-pS6, mouse anti-S6, rabbit anti-pERK, rabbit anti-ERK, rabbit anti-pan-RAS (Cell Signaling, Beverly, MA, USA). Mouse anti-GAPDH (HyTest, Turku, Finland) or mouse anti-β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, donkey anti-goat IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology Inc.), and Goat anti-mouse IgG1 or Goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL, USA) conjugated to horseradish peroxidase were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Also, donkey anti-mouse IgG or donkey anti-rabbit (LI-COR Biotechnology, Bad Homburg, Germany) labeled with IRDye infrared dyes were used for detection. Representative blots of at least two independent experiments are shown.

Schott C., Graab U., Cuvelier N., Hahn H, & Fulda S. (2015). Oncogenic RAS Mutants Confer Resistance of RMS13 Rhabdomyosarcoma Cells to Oxidative Stress-Induced Ferroptotic Cell Death. Frontiers in Oncology, 5, 131.

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Immunohistochemical Profiling of Neural Cells

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Immunohistochemical labeling of embryonic brain sections or dissociated neural cells was performed as described previously (Kim et al., 2006 (link)). The following primary antibodies were used: rabbit anti-mTOR (Cell Signaling), rabbit anti-4EBP1 (Cell Signaling), mouse anti-S6 (Cell Signaling), rabbit anti-TSC2 (Cell Signaling), rabbit anti-phospho-mTOR (Cell Signaling), rabbit anti-phospho-4EBP1 (Cell Signaling), rabbit anti-phospho-S6 (Cell Signaling), rabbit anti-phospho-S6K (Cell Signaling), rabbit anti-phospho-histone H3 (Upstate Biotech), mouse anti-BrdU (Sigma), rabbit anti-Ki67 (Covance), rabbit anti-Sox2 (Chemicon), rabbit anti-Tbr1 (Chemicon), rabbit anti-Cux1 (Santa Cruz), goat anti-Brn1 (Novus Biologicals), rabbit anti-Tbr2 (Abcam), mouse anti-Tuj1 (Sigma). Appropriate secondary antibodies conjugated with Alexa Fluor dyes (Invitrogen) were used to detect primary antibodies.

Ka M., Condorelli G., Woodgett J.R, & Kim W.Y. (2014). mTOR regulates brain morphogenesis by mediating GSK3 signaling. Development (Cambridge, England), 141(21), 4076-4086.

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Western Blot Analysis of Apoptosis Regulators

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Western blot analysis was performed as described previously [52 (link)] using the following antibodies: mouse anti-caspase-8, mouse anti-NOXA, rat anti-BMF (Alexis Biochemicals, Grünberg, Germany), mouse anti-AKT, mouse anti-BCL-2, mouse anti-BAX, rabbit anti-BAK (BD Bioscience), rabbit anti-caspase-3, mouse anti-caspase-9, rabbit anti-pAKT, rabbit anti-p4E-BP1, rabbit anti-4E-BP1, rabbit anti-pS6, mouse anti-S6, (Cell Signaling, Beverly, MA), rabbit anti-MCL-1 (Stressgene Bioreagents, Victoria, BC). Mouse anti-GAPDH (HyTest, Turku, Finland) or mouse anti-β-Actin (Sigma) were used as loading controls. Goat anti-mouse IgG, donkey anti-goat IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA)) and Goat anti-mouse IgG1 or Goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL) conjugated to horseradish peroxidase were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Representative blots of at least two independent experiments are shown.

Graab U., Hahn H, & Fulda S. (2015). Identification of a novel synthetic lethality of combined inhibition of hedgehog and PI3K signaling in rhabdomyosarcoma. Oncotarget, 6(11), 8722-8735.

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Antibody Characterization and Validation

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Polyclonal rabbit antibodies were raised against the synthetic peptide NH₂-NPGLDARIPSLAELEC-CONH₂ of human B⁰AT1 and further affinity purified (Pineda, Berlin, Germany). Mouse anti-TMEM27 (Abnova, Taipei, Taiwan), mouse anti-EGFP (Clontech), rabbit anti-4E-BP1 (Cell Signaling, Danvers, MA, United States), rabbit anti-phospho-4E-BP1 (Thr70) (Cell Signaling), rabbit anti-eIF2α (Cell Signaling), rabbit anti-phospho-eIF2α (Ser51) (Cell Signaling), rabbit anti-ZO-1 (Life Technologies) and mouse anti-β-actin (Sigma-Aldrich) were used according to the manufacturers' instructions. Horse radish peroxidase goat anti-rabbit IgG and alkaline phosphatase goat anti-mouse IgG secondary antibodies were purchased from Promega (Dübendorf, Switzerland).

Torrente M., Guetg A., Sass J.O., Arps L., Ruckstuhl L., Camargo S.M, & Verrey F. (2014). Amino Acids Regulate Transgene Expression in MDCK Cells. PLoS ONE, 9(5), e96823.

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Antibody Profiling for Apoptosis Signaling

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Antibodies used for immunoblotting analyses in this study are listed below: rabbit anti-MCL-1 (1:500, ab32087), rabbit anti-BAX (1:1000, ab32503) were obtained from Abcam; mouse anti-BCL-2 (1:1000, #15071), rabbit anti-BIM (1:1000, #2933), rabbit anti-BCL-XL (1:1000, #2762), rabbit anti-BAK (1:1000, #12105), rabbit anti-AKT (1:1000, #4691), rabbit anti-p-AKT (1:1000, #4060), rabbit anti-mTOR (1:1000, #2983), rabbit anti-p-mTOR (1:1000, #5536), rabbit anti-p-4EBP1 (1:1000, #2855), rabbit anti-4EBP1 (1:1500, #9644), rabbit anti-FOXO3a (1:1000, #12829), rabbit anti-p-FOXO3a (1:1000, #9466) rabbit anti-cleaved PARP (1:1000, #5625) and rabbit anti-cleaved Caspase-3 (1:1000, #9664) were purchased from Cell Signaling Technology; mouse anti-β-actin (1:1000, sc-47778), mouse anti-GAPDH (1:1000, sc-32233) and rabbit anti-PUMA (1:1000, sc-28226) were from Santa Cruz Biotechnology; rabbit anti-p-4EBP1 (1:500, NB100-81769, used in Fig. 1b) and mouse anti-4EBP1 (1:1000, NBP1-47366, used in Fig. 1b) were obtained from Novus Biologicals. The compounds of BH3 mimetics (ABT263 and ABT199) and mTOR inhibitors (BEZ235, AZD8055, and Temsirolimus) were from AbMole Bioscience.

Li H., Liu L., Chang H., Zou Z, & Xing D. (2018). Downregulation of MCL-1 and upregulation of PUMA using mTOR inhibitors enhance antitumor efficacy of BH3 mimetics in triple-negative breast cancer. Cell Death & Disease, 9(2), 137.

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Immunochemical Analysis of Cellular Signaling Proteins

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Rabbit anti-S6, rabbit anti-phospho-S6 (ser235, 236), rabbit anti-4EBP-1, rabbit anti-phospho-4EBP-1 (Thr37, 46), rabbit anti-β-Catenin (species cross-reactivity: human, mouse, and rat) and mouse anti-proliferating cell nuclear antigen (PCNA) were from Cell Signaling Technology (Beverly, MA). Rabbit anti-α-Amylase was from Sigma Chemical Co. (St. Louis, MO). Mouse anti-CA19-9 was from Origene (Beijing, China). Goat anti-insulin A (C-12) (species cross-reactivity: human, mouse, and rat), Goat anti-glucagon (N-17) (species cross-reactivity: human, mouse, and rat), rabbit anti-somatostatin (FL-116) (species cross-reactivity: human, mouse, and rat), goat anti-rabbit fluoresceinisothiocyanate-conjugated IgG, donkey anti-goat fluoresceinisothiocyanate- conjugated IgG, donkey anti-goat Texas Red-conjugated IgG, goat anti-mouse Texas Red-conjugated IgG, and chicken anti-rabbit fluoresceinisothiocyanate-conjugated IgG and rapamycin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Dimethylsulfoxide was from Sigma Chemical Co. (St. Louis, MO). Aprotinin was purchased from Amersham Biosciences (Pittsburgh, PA). IRDye-conjugated affinity purified anti-rabbit, anti-mouse IgGs were purchased from Rockland (Gilbertsville, PA).

Ding L., Han L., Li Y., Zhao J., He P, & Zhang W. (2014). Neurogenin 3–Directed Cre Deletion of Tsc1 Gene Causes Pancreatic Acinar Carcinoma. Neoplasia (New York, N.Y.), 16(11), 909-917.

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Protein Isolation and Western Blot Analysis

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Proteins of cells, and frozen prostate and detrusor tissues were isolated according to a previously described method¹⁹ (link). Primary antibodies for Western blot analyses included rabbit anti-phospho-cofilin (Ser3; 77G2, #3313), rabbit cofilin (D3F9, #5175S), rabbit anti-phospho-LIMK1 (Thr508)/LIMK2 (Thr505, #3841), rabbit anti-myosin-binding subunit (MYPT1, #2634), rabbit anti-myosin light chain (MLC) 2 (#3672), rabbit anti-phospho-myosin light chain 2 (Ser19; #3671), rabbit anti-phospho-eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, Thr37/46; #9459), rabbit anti-phospho-4E-BP1 (Ser65; #9451), rabbit anti-4E-BP1 (#9452) (Cell Signaling Technology, USA), mouse anti-calponin 1/2/3 (sc-136987), mouse anti-vimentin (sc-6260), mouse monoclonal anti-GAPDH (sc-32233) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-LIMK1 (ab81046), rabbit anti-LIMK2 (ab45165) (Abcam, Cambridge, UK). Detection was continued using secondary antibodies IRDye® 800CW goat anti-mouse (or rabbit) IgG and IRDye® 680RD goat anti-mouse (or rabbit) IgG (LI-COR). The bands were detected by using Odyssey® Clx Imaging Systems and quantified with respect to GAPDH using ImageJ software.

Yu Q., Wu C., Chen Y., Li B., Wang R., Huang R., Li X., Gu D., Wang X., Duan X., Li S., Liu Y., Wu W., Hennenberg M, & Zeng G. (2021). Inhibition of LIM kinase reduces contraction and proliferation in bladder smooth muscle. Acta Pharmaceutica Sinica. B, 11(7), 1914-1930.

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Western Blot Analysis of Breast Cancer Cells

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MCF-7 and MDA-MB-231 cells were lyzed in MPER (Thermo Scientific, Bleiswijk, The Netherlands) and diluted 1:1 with SDS sample buffer (4% SDS, 20% glycerol, 0.5 mol/l Tris-HCl (pH 6.8), 0.002% bromophenol blue). Lysates were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated overnight at 4 °C and probed with the following antibodies: rabbit-anti-AKT, rabbit-anti-pAKT (Thr308), rabbit-anti-S6, rabbit-anti-pS6, rabbit-anti-4EBP1 (all Cell Signaling Technologies, Leiden, The Netherlands) in a 1:1000 dilution or anti-HIF1α (BD Biosciences, Breda, The Netherlands) and mouse-anti-actin (MP Biomedicals, Santa Ana, USA) in a 1:10,000 dilution. Primary antibodies were stained using HRP-coupled goat anti-rabbit or rabbit anti-mouse IgG and developed with Lumi-Light (Roche, Almere, The Netherlands). Images were captured with the ChemiDoc MP imaging system (Bio-Rad, Veenendaal, The Netherlands) and Image Lab Software.

Ariaans G., Jalving M., Vries E.G, & Jong S.D. (2017). Anti-tumor effects of everolimus and metformin are complementary and glucose-dependent in breast cancer cells. BMC Cancer, 17, 232.

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Quantification and Western Blot Analysis of Protein Targets

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The freshly isolated islets were lysed, quantified, blotted and developed as described before21 (link). Primary antibodies are listed as following: rabbit anti-RAPTOR (1:1,000, Cell Signaling), rabbit anti-PS6 (Ser240/244) (1: 1,000, Cell Signaling), rabbit anti-4E-BP1 (1: 1,000, Cell Signaling), rabbit anti-PS6K (Thr389) (1:1,000, Cell Signaling), mouse anti-DNMT3A (1: 2,000, Novus Biologicals), rabbit anti-RPL7 (1: 1,000, Bethyl), rabbit anti-RPL26 (1: 1,000, Bethyl) and rabbit anti-LDHA (1: 1,000 Abcam), rabbit anti-PGC1a (1: 1,000 Abcam), mouse anti-TUBULIN (1: 20,000, Sigma-Aldrich). TUBULIN was used as an internal control to normalized band intensity.

Ni Q., Gu Y., Xie Y., Yin Q., Zhang H., Nie A., Li W., Wang Y., Ning G., Wang W, & Wang Q. (2017). Raptor regulates functional maturation of murine beta cells. Nature Communications, 8, 15755.

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Antibody Selection for Neurodegenerative Research

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Previously described procedures were followed for these experiments (43 (link), 44 (link)). The antibodies used were rabbit anti-AGAT (orb247515, Biorbyt); mouse anti-GAMT (sc-398936, Santa Cruz Biotechnology); mouse anti–synaptotagmin 1/2 (105011, Synaptic Systems); rabbit anti–PSD-95 (AB9708, Millipore); rabbit anti-Homer1 (ab211415, Abcam); rabbit anti–p-AMPK (Thr172) (2531S, Cell Signaling Technology); rabbit anti–p-UL K (Ser317) (12753, Cell Signaling Technology); rabbit anti-p62 rabbit (8025S, Cell Signaling Technology); rabbit anti–p-mTOR (Ser2448) (2971S, Cell Signaling Technology); rabbit anti-mTOR (2972S, Cell Signaling Technology); rabbit anti–p70 S6 kinase (Thr389) (9205S, Cell Signaling Technology); rabbit anti–p-S6 (Ser240/244) (5364S, Cell Signaling Technology); rabbit anti-S6 (2217S, Cell Signaling Technology); rabbit anti–p-4E-BP1(Thr37/46) (2855S, Cell Signaling Technology); rabbit anti–4E-BP1 (9644S, Cell Signaling Technology); rabbit anti–p-ULK (Ser757) (6888S, Cell Signaling Technology); rabbit anti-GAPDH (5174S, Cell Signaling Technology); rabbit anti-LC3B (ab48394, Abcam); and rabbit anti–cleaved caspase-3 (Asp175) (9661S, Cell Signaling Technology).

Chen H.R., Zhang-Brotzge X., Morozov Y.M., Li Y., Wang S., Zhang H.H., Kuan I.S., Fugate E.M., Mao H., Sun Y.Y., Rakic P., Lindquist D.M., DeGrauw T, & Kuan C.Y. (2021). Creatine transporter deficiency impairs stress adaptation and brain energetics homeostasis. JCI Insight, 6(17), e140173.

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