Reagents used in this study include the following: Methylthiazolyldiphenyl-tetrazolium bromide (MTT)(S6821), PKI-402(S2739), cycloheximide (CHX)(NSC-185) and Thapsigargin (Tg) (S7895)were commercially sourced from Selleckchem (Houston, TX, USA), anti-G3BP1 (13057-2-AP), anti-eIF2α (11233-1-AP), anti-4EBP1 (60246-1-Ig), anti-caspase-9(66169-1-Ig), anti-Bcl-2 (26593-1-AP), anti-Bax (50599-2-Ig), anti-Hsp60 (15282-1-AP), anti-Lonp (66043-1-Ig), anti-Clpp (15698-1-AP), anti-Trap1 (10325-1-AP), anti-Actin(HRP-60008), anti-VDAC (10866-1-AP) (Proteintech, Chicago, IL, USA), anti-YB-1(sc-101198), anti-ATF5(sc-377168), anti-p-Akt(Ser473) (sc-101629)(Santa Cruz, CA, USA), p- eIF2α(Ser51)(3398), p-4EBP1(Thr37/46)(2855), p-Akt(Thr308) (13038), p-P70S6K(9204) (Cell Signaling Technology, USA).
Anti 4ebp1
Manufactured by Proteintech
Sourced in United States
The Anti-4EBP1 is a laboratory reagent used for the detection and quantification of 4EBP1 protein in various biological samples. 4EBP1 is a key regulator of protein translation and cell growth. The antibody can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and ELISA, to study the expression and localization of 4EBP1 in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Proteintech (2 mentions)
Cycloheximide (chx), by Selleck Chemicals (1 mentions)
Thapsigargin, by Selleck Chemicals (1 mentions)
Anti p akt ser473, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 9, by Proteintech (1 mentions)
Anti bcl 2, by Proteintech (1 mentions)
Anti clpp, by Proteintech (1 mentions)
Anti vdac, by Proteintech (1 mentions)
Anti hsp60, by Proteintech (1 mentions)
Anti atf5, by Santa Cruz Biotechnology (1 mentions)
Pki 402, by Selleck Chemicals (1 mentions)
Anti eif2α, by Proteintech (1 mentions)
P eif2α, by Cell Signaling Technology (1 mentions)
P 4ebp1, by Cell Signaling Technology (1 mentions)
Anti bax, by Proteintech (1 mentions)
Ab54503, by Abcam (1 mentions)
Ab205719, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Protease phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
P mtor, by Proteintech (1 mentions)
5 protocols using anti 4ebp1
1
Investigating Cellular Stress Responses
2
Investigating mTOR Pathway Regulation in AML
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AML cells were cultured in complete medium containing 10% serum, stimulated with 25 ng/ml IL6 for 30 min. Cells were harvested, washed with 1 × PBS and lysated with 1 × RIPA lysis buffer containing protease/phosphatase inhibitors (Thermo Fisher #78,441). The lysates were sonicated and centrifuged at 14,000 g for 15min. Western blot analysis was performed with the following primary antibodies, including anti-4EBP1 (CST#9644S), p-4EBP1 (CST#2855P), p-mTOR (CST#5536S), mTOR (CST#2983S), p-STAT3 (CST#9145S), STAT3 (CST#4904S), p-P70S6K (CST#9205S), P70S6K (CST#35,708), CDK6 (Proteintech#14,052), CCND1 (Abcam#ab54503), GAPDH (CST#5174), and HRP conjugated secondary antibodies (Abcam#ab205718, Abcam#ab205719). Detection was conducted using a chemiluminescence substrate (Omics Bio), and images were acquired using ImageQuantTM LAS 4,000 camera and quantified using lmageJ software version1.53 (NIH, Bethesda, MD, USA). To examine whether the phosphorylated protein levels of mTOR and its downstream effectors (P70S6K and 4EBP1) were significantly inhibited, we normalized the levels of phosphorylated protein to the corresponding total protein. Uncropped images for the blots were provided in Additional file 12 .
3
Characterization of Breast Cancer and Control Cell Lines
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Human breast cancer cell lines MDA-MB-468, BT474, SKBR-3 and normal breast epithelial cell line Hs578Bs were purchased from the Cell Center of Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Human breast cancer cell lines MCF-7, MDA-MB-231 and human embryonic kidney cell line HEK293T were kept in our laboratory. EBSS (Hyclone) starvation was carried out to switch the culture medium from the complete medium to EBSS medium. Commercially available antibodies and dilutions used are as follows: anti-ITM2A (Proteintech, cat: 18306–1-AP, 1:1000 dilution), anti-GAPDH (Proteintech, cat: 60004–1-Ig, 1:1000 dilution), anti-LC3 (Cell Signaling Technology, cat: 12741, 1:1000 dilution), anti-P62/SQSTM1 (Boster, cat: BM4385, 1:1000 dilution), anti-Rabbit Flag (EMD Millipore, cat: PM020A, 1:1000 dilution), anti-pAMPK-T172 (Cell Signaling Technology, cat: 2535, 1:1000 dilution), anti-AMPK (Boster, cat: A30453, 1:500 dilution), anti-p4EBP1-T37/46 (Cell Signaling Technology, cat: 2855, 1:1000 dilution), anti-4EBP1 (Proteintech, cat: 60246–1-Ig, 1:1000 dilution). anti-phospho-MBP (EMD Millipore, cat: 05–429, 1:1000 dilution), anti-MBP (Proteintech, cat: 10458–1-AP, 1:1000 dilution), anti-HUNK (Invitrogen, cat: PA5–28765, 1:1000 dilution).
4
Protein Expression Analysis of EDL Muscle
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The total protein of the EDL was extracted and quantified using a protein assay kit (Thermo Fisher Scientific, USA). Protein samples were loaded onto 15 wells (20 μg total protein per well) of 4%–12% Bis-Tris or 3%–8% Tris Acetate gradient gels (NW04125/EA03755, Invitrogen, USA). After separation by electrophoresis, the proteins were transferred onto a nitrocellulose Regular Stack (IB23001, Invitrogen). Using Odyssey Blocking buffer (LI-COR) as the blocking reagent, the target proteins were blocked and probed overnight at 4°C with anti-mTOR (Abcam, ab134903), anti-p-mTOR (Cell Signaling Technology/CST, 5536T), anti-4E-BP1 (Proteintech, 60246-1-Ig), anti-p-4E-BP1 (CST, 2855), anti-FoxO1 (Santa, sc-374427), anti-p-FoxO1 (Abcam, ab131339), anti-ubiquitin (Santa, sc-8017), anti-NF-κB (CST, 8242), anti-ASC (Proteintech, 10500-1-AP), anti-Caspase1 (CST, 2225), and anti-β-tubulin (Proteintech, 10094-1-AP) antibodies. After washing three times (10 min per wash time) with Tris-buffered saline (TBS) containing Tween-20, the membranes were incubated with goat anti-rabbit or anti-mouse IgG secondary antibodies (LI-COR, 926-68071/926-32210) at 25°C for 1 h. Next, the membranes were washed twice with TBS and signals were detected using a near-infrared spectroscopy detection system (Odyssey CLX, LI-COR). All bands were analyzed semi-quantitatively using Image Studio Ver 5.2.
5
Western Blot Analysis of Signaling Proteins
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Western blot was performed as previously described [16] . Briefly, whole-cell lysates were collected, and protein concentrations were quantified by a BCA Protein Assay Kit (AR0146, Boster Biological Technology Ltd, Wuhan, China). The immunoblots were probed with primary antibodies overnight at 4 °Cfollowed by secondary antibodies at room temperature for 1 h. The following primary antibodies were used according to the manufacturer's instructions: anti-phospho-FGFR2 (1:1000, 3471, Cell Signaling Technology, USA), anti-FGFR2 (1:1000, TA503137, OriGene, USA), anti-TSP4 (AP19723b, Abgent, USA), anti-phospho-Akt (1:1000, 4060, Cell Signaling Technology, USA), anti-Akt (1:1000, 4691, Cell Signaling Technology, USA), anti-phospho-mTOR (1:1000, 5536, Cell Signaling Technology, USA), anti-mTOR (1:1000, 20657-1-AP, Proteintech, USA), anti-phospho-p70S6K (1:1000, 9234, Cell Signaling Technology, USA), anti-p70S6K (1:1000, 14485-1-AP, Proteintech, USA), anti-phospho-4EBP1 (1:1000, 2855, Cell Signaling Technology, USA), anti-4EBP1 (1:1000, 60246-1-lg, Proteintech, USA), and anti-GAPDH (1:5000, 60004-1-lg, Proteintech, USA). The blots were visualized using an enhanced chemiluminescencedetection kit (Thermo Fisher Scientific, USA).
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