Proteins were extracted from RMCs using RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM PMSF, and protease cocktail at 1 μg/mL). Protein concentrations were determined using a BCA kit (Thermo Scientific, Rockford, AL, USA). Equal amounts of protein (60 µg) were separated by SDS-PAGE on a 6%, 10%, or 12% acrylamide gel and then transferred onto a nitrocellulose membrane. After the membrane had been blocked with non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature, it was probed with the following primary antibodies overnight at 4°C: anti-VDR (catalogue no.: ab109234, Abcam, USA), anti-DDIT4 (catalogue no.: NBP1–77321, Novus Biologicals, USA), anti-TSC1 (catalogue no.: 6935S, Cell Signaling Technology, USA), anti-TSC2 (catalogue no.: 4308P, Cell Signaling Technology), anti-Rheb (catalogue no.: 13879S, Cell Signaling Technology), anti-mTOR (catalogue no.: Ab51044, Abcam), anti-4E-BP1 (catalogue no.: ab2606, Abcam), and anti-p70S6K (catalogue no.: ab32359, Abcam). After extensive washing, the membranes were incubated with Dylight anti-rabbit IgG secondary antibody. The antigens were visualized using the Odyssey infrared imaging system (LI-COR Biotechnology, Nebraska, USA). The results were expressed as the relative intensity (RI) intensity (adjusted to that of β-actin) of each band.
Anti 4e bp1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-4E-BP1 is a primary antibody that recognizes the 4E-BP1 protein. 4E-BP1 is a key regulator of protein synthesis and cell growth. This antibody can be used to detect and study the 4E-BP1 protein in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti p70s6k, by Abcam (4 mentions)
Anti mtor, by Abcam (4 mentions)
Anti gapdh, by Abcam (4 mentions)
Anti p 4e bp1, by Abcam (3 mentions)
Anti p p70s6k, by Cell Signaling Technology (2 mentions)
Anti p p70s6k, by Abcam (2 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Anti tsc2, by Cell Signaling Technology (1 mentions)
Anti vdr, by Abcam (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Anti rheb, by Cell Signaling Technology (1 mentions)
Anti tsc1, by Cell Signaling Technology (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Anti mdr1, by Abcam (1 mentions)
Anti p 4ebp1, by Cell Signaling Technology (1 mentions)
Anti α tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Biotinylated rabbit anti mouse, by Agilent Technologies (1 mentions)
Biotinylated goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti 4e bp1
1
Western Blot Analysis of mTOR Pathway
2
Western Blot Analysis of mTOR Pathway
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Western blot analysis was performed as described previously [8 (link)]. The following antibodies were used: anti-mTOR (1:400 dilution; Cell Signaling, Natick, MA, USA), anti-p-mTOR (2481 and 2448) (1:1000; Cell Signaling), anti-p-4EBP1 (1:1000; Cell Signaling), anti-p-p70s6k (1:1000; Cell Signaling), anti-4EBP1 (1:1000; Abcam, Cambridge, UK), anti-P70s6K (1:1000; Abcam), anti-MDR1 (1:1000; Abcam), anti-GAPDH (1:2000; Abcam), and anti-HRP (1:2000; Cell Signaling).
3
Quantitative Western Blot Analysis
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Western blot was carried out as previously described49 (link). The primary antibodies used were mouse monoclonal anti-glutamine synthetase (Abcam, Cambridge, UK), anti-β-actin antibody (Sigma-Aldrich, Saint-Louis, Missouri, USA), anti-α-tubulin antibody (Santa Cruz, Dallas, Texas USA.), rabbit polyclonal anti-asparagine synthetase (Abcam), anti-4E-BP1, anti-4E-BP1-pT70, anti-Erk1/2, anti-Erk1/2-pT202/Y204, anti-elF-2α-pS51, anti-elF-2α (Ozyme, Saint Quentin Yvelines, France), and anti-BCL2A1 antibody (Abcam). Secondary antibodies were as follows: biotinylated rabbit-anti mouse (Dako, Courtaboeuf, France) and biotinylated goat anti-rabbit (Invitrogen, Carlsbad, CA, USA). Detection was performed using Luminata Forte Western HRP substrate (Millipore Corporation, Billerica, MA). Quantifications were carried out by densitometric analysis using Kodak software as previously described49 (link). β-actin or α-tubulin was used as loading control.
4
Molecular Mechanisms of AMPK Regulation
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PF‐06409577 (#M8095, Abmole, Shanghai, China), Forskolin (FSK; #F6886, Sigma, Shanghai, China) and 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP; #B5386, Sigma) were dissolved in 100% DMSO as 100 mm stock solutions and stored at −20 °C. DMSO (#D2650) and 3‐Isobutyl‐1‐methylxanthine (IBMX; #I5879‐1G) were from Sigma. Anti‐AMPK (1 : 1000, #2532), anti‐p‐AMPK (1 : 1000, #2535), anti‐AKT (1 : 1000, #9272), anti‐p‐AKT (1 : 1000, #9271), anti‐p70S6K (1 : 1000, #2708) and anti‐p‐p70S6K (1 : 1000, #9234) were from Cell Signaling Technology (CST, Shanghai, China). Anti‐4EBP1 (1 : 1000, #ab32024), anti‐p‐4EBP1 (1 : 1000, #ab75767), anti‐GAPDH (1 : 1000, #ab9585) and goat anti‐rabbit IgG (1 : 5000, #ab216773) were from Abcam (Shanghai, China). CFTRInh‐172 (#SF9110) was from Beyotime Biotechnology (Shanghai, China). PEG300 (#HY‐Y0873) was from MCE (Shanghai, China). Tween80 (#1716) was from Biofroxx (Guangzhou, China). Other chemicals were of analytical grade and obtained from standard commercial sources.
5
Western Blot Analysis of MELK and mTOR Signaling
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Cells were lysed in lysis buffer including 0.2% protease inhibitor cocktail III (Calbiochem, San Diego, CA; La Jolla, CA, USA). After homogenization, the cell lysates were incubated on ice for 30 min and centrifuged at 12,000 g for 10 min. The amount of total protein was measured by protein assay kit (Bio-Rad, Hercules, CA, USA), and the proteins were then mixed with SDS sample buffer and boiled for 5 min before loading into a 10% or 8% SDS-PAGE gel (Bio-Rad, Hercules, CA, USA). The proteins were transferred onto nitrocellulose membrane after electrophoresis. The blots were blocked and then incubated with primary antibody followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. Immunoreactive bands were visualized by enzyme-linked chemiluminescence with an ECL kit. The primary antibodies were as follows: anti-MELK (ab108529), anti-α-tubulin (ab7291), and anti-mTOR (ab2732) from Abcam (Cambridge, UK); anti-4E-BP1 (#9644), anti-p-4E-BP1 (T37/46) (#2855), anti-S6 (#2317), anti-p-S6 (S235/236) (#4858), anti-p-PRAS40 (Thr246) (#13175), anti-p-PRAS40 (Ser183) (#5936), and anti-raptor (#2280) from Cell Signaling Technology (Danvers, MA, USA); and anti-Flag from Sigma (#3165, St Louis, MO, USA). The immune complex was detected using HRP-conjugated secondary antibodies (ZSGB-BIO, Beijing, China). Rapamycin was purchased from Sigma (Solon, OH, USA).
6
Protein Expression and Signaling in Cellular Responses
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LPS from Escherichia coli O127:B8 was purchased from Sigma-Aldrich; Merch KGaA. The mTOR activator 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-one (3BDO; cat. no. S8317) and the AMPK inhibitor compound C (cat. no. S7306) were obtained from Selleck Chemicals. The primary antibodies used for western blotting were the following: Anti-AMPKα (1:1,000; cat. no. ab32047; Abcam), anti-phosphorylated-AMPKαThr172 (p-AMPKα; 1:1,000; cat. no. ab133448; Abcam), anti-4E-BP1(1:2,000; cat. no. ab32024; Abcam), anti-phosphorylated-4E-BP1 (p-4E-BP1; 1:1,000; cat. no. ab278686; Abcam), anti-PFKFB3 (1:2,000; cat. no. ab218121; Abcam), anti-mTOR (1:1,000, cat. no. 2983; Cell Signaling Technology, Inc.), anti-phosphorylated-mTOR (p-mTOR, 1:1,000; cat. no. 2971; Cell Signaling Technology, Inc.), anti-collagen I (1:1,000; cat. no. ab260043; Abcam), anti-α-smooth muscle actin (α-SMA, 1:1,000; cat. no. 19245; Cell Signaling Technology, Inc.), anti-collagen III (1:1,000; cat. no. ab184993; Abcam) and anti-GAPDH (1:2,000; cat. no. 5174; Cell Signaling Technology, Inc.). Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:2,000; cat. no. A0208; Beyotime Institute of Biotechnology) were used in this study.
7
Proteomic Analysis of BMSC Proteins
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Proteins were extracted from more than 106 harvested BMSCs according to the kit protocol (KeyGen Biotech, China). [46 (link)] BMSCs were digested in a lysis buffer cocktail and centrifuged at 12000 g for 15 minutes at 4 °C to get the supernatant as the total protein. 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used for electrophoresis of protein (20 μg) with loading buffer and then blotted to a polyvinylidene fluoride (PVDF) membrane (Millipore Co., USA). Membranes were blocked with 5% skimmed milk in Tris-buffered saline tween 20, TBST, and then incubated overnight at 4°C with primary antibodies; anti-IHH, anti-P53, anti-P16, anti-PI3K, anti-p-PI3K, anti-Akt 1, anti-p-Akt 1, anti-NF-κB, anti-p-NF-κB-p65, anti-STAT3, anti-p-STAT3, anti-4EBP1, anti-p- 4EBP1, anti-p70S6K, anti-p-p70S6K and anti-β-actin (1:1000), all from Abcam, USA. The membranes were incubated for 1hour at RT with secondary antibody conjugated with horseradish peroxidase. Finally, TBST-washed membranes were treated with enhanced chemiluminescent (ECL) for detection (Bio-Rad, USA). Image Lab detection system (Bio-Rad, USA) was used for protein bands imaging and analysis.
8
Elucidating Autophagy Regulation by TEOA in Cells
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TEOA was obtained from college of pharmacy, Zhejiang University (Zhejiang, China) and dissolved in DMSO. N-acetylcysteine (NAC), Chloroquine (CQ), 3-Methyladenine (3MA) and Rapamycin were purchased from Medchem Express (MCE, United States). DAPI was purchased from Beyotime (Shanghai, China). The following primary antibodies were used: anti-GAPDH (Abcam ab181602) (1:2,000), anti-Catalase (Abcam ab76024) (1:1,000), anti-MnSOD (Abcam ab68155) (1:2,000), anti-LC3 (Sigma L7543) (1:1,000), anti-p-mTOR (Abcam ab109268) (1:2,000), anti-mTOR (Abcam ab134903) (1:1,000), anti-p-p70S6K (Abcam ab131436) (1:1,000), anti-p70S6K (Abcam ab32529) (1:1,000), anti-p-S6 (CST 4858S) (1:2,000), anti-S6 (CST 2217s) (1:1,000), anti-p-4EBP1 (CST 9459S) (1:1,000), anti-4EBP 1 (CST 9644S) (1:1,000), anti-ATG5 (Abcam ab68155) (1:2,000). The related HRP-conjugated secondary antibody was purchased from Beyotime (Shanghai, China).
9
Molecular Mechanisms of Rapamycin and SC75741
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Rapamycin (Gene Operation, Ann Arbor, MI, USA) was dissolved in ethanol (Sigma-Aldrich, Inc., USA) to a stock concentration of 50 mg/ml and stored at -20°C, and was diluted to target final concentrations with culture medium before use. SC75741 (America Selleck Biotechnology Co., Ltd Houston Texas USA) was dissolved in DMSO to a stock concentration of 50 mM, and was diluted to target final concentrations with culture medium before use. The concentration of ethanol in the final solution did not exceed 0.5% (v/v) in any experiment. The following primary antibodies were purchased from Cell Signaling Technology, Inc. (Beverley, MA, USA): anti-NF-κB p65, anti-phospho-NF-κB p65 (Ser536), anti-p-4EBP1 (Thr37/46), anti-p-S6 (Ser240/244), and anti-Raptor. anti-S6 primary antibody was purchased from Santa Cruz Biotechnology, Inc. (CA, USA). anti-4EBP1, anti-p-mTOR (Ser2448), and anti-mTOR were purchased from Abcam (plc 330 Cambridge Science Park, Cambridge, UK). Anti-β-actin primary antibodies were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). ECL Anti-Rabbit IgG-HRP and ECL Anti-Mouse IgG-HRP were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK).
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