Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F-12 (DMEM/F-12), fetal bovine serum (FBS), penicillin–streptomycin (PS), and customized DMEM were purchased from Gibco BRL company (Waltham, MA, USA). Antibodies against mammalian target of rapamycin complex 1 (mTORC1, catalog no. #2972), phosphorylated (p)-mTORC1 (catalog no. #2971), eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1, catalog no. #9452), p-4E-BP1 (catalog no. #13396), ribosomal protein S6 kinase (p70S6K, catalog no. #9202), p-p70S6K (catalog no. #9234), S6 (catalog no. #2217), p-S6 (catalog no. #2211), and AMP-activated protein kinase (AMPK, catalog no. #2532), and p-AMPK (catalog no. #2535) were products of Cell Signaling Technology (Beverly, MA, USA). Antibody against proline dehydrogenase (PRODH, catalog no. ab203875) was procured from Abcam (Cambridge, UK). Antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, catalog no. sc-59540) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Peroxidase-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were obtained from Huaxingbio Biotechnology Co. (Beijing, China). 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Unless otherwise indicated, all other chemicals used in this study were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Mtorc1
Manufactured by Cell Signaling Technology
Sourced in United States
MTORC1 is a protein complex that plays a central role in regulating cell growth, metabolism, and proliferation. It serves as a sensor of cellular nutrients and energy levels, and coordinates various cellular processes accordingly.
Automatically generated - may contain errors
Lab products found in correlation
P mtorc1, by Cell Signaling Technology (6 mentions)
4e bp1, by Cell Signaling Technology (3 mentions)
P 4ebp1, by Cell Signaling Technology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
P ampk, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Ez ecl, by Sartorius (2 mentions)
P trkb, by Santa Cruz Biotechnology (2 mentions)
Isoflurane, by Merck Group (2 mentions)
P gsk3β, by Santa Cruz Biotechnology (2 mentions)
Phosphatase and tensin homolog pten, by Cell Signaling Technology (2 mentions)
Mtorc2, by Cell Signaling Technology (2 mentions)
P mtorc2, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
15 protocols using mtorc1
1
Signaling Pathways in Cellular Response
2
Molecular Profiling of mPFC in Rats
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Animals were sacrificed and brains were rapidly removed and frozen in liquid nitrogen. mPFC was dissected out bilaterally using a brain mold on ice. Homogenates of the dissected tissue were lysed using lysis buffer (Beyotime Biotechnology, China) containing protease inhibitors (Sigma, USA) and phosphorylase inhibitors (Sigma, USA). Electrophoresis was performed on 4–15% Mini-PROTEAN TGX precast gels (Bio-Rad, USA). Primary antibodies used included anti-BDNF (1:500, Abcam, Cat GR3227037-2, USA), -M2-AChR (1:500, Abcam, Cat GR50911-14, USA), -mTORC1 (1:500, Cell Signaling Technology, Cat 2983S, USA), -phospho-mTORC (1:500, Cell Signaling Technology, Cat 5536S, USA) and -β-Actin (1:1,000, Santa Cruz, Cat SC47778, USA). Secondary antibodies used included HRP anti-rabbit antibody (1:5,000, Beyotime, China) and HRP anti-mouse antibody (1:5,000, Beyotime, China). Bands were detected using a chemiluminescence imaging system (Bio-Rad, USA). Image J (NIH, USA) software was used to analyze band densitometry.
3
Chondrocyte Apoptosis and Autophagy Regulation
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Eicosapentaenoic Acid (purity, ≥97%), 3-Methyladenine (3-MA, a PI3K inhibitor) and Dorsomorphin (Compound C, an AMPK inhibitor) were purchased from MedChemExpress (NJ, United States). Type II collagenase, TBHP, and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (St Louis, MO, United States). Primary antibodies for cleaved-caspase 3 (C-C3), Beclin-1, LC3B, p-mTORC1, mTORC1, BCL-2, AMPK and p-AMPK were procured from Cell Signaling Technologies (Danvers, MA, United States). Antibodies against BAX, BCL-2, Collagen II, MMP13 and P62 were purchased from Abcam (Cambridge, United Kindom). Antibodies against p-PERK, PERK, p-eIF2α, eIF2α, CHOP, ADAMTS5, Aggrecan, and ATG5 were purchased from ABclonal (WH, CHINA). Antibodies against ATF4, GRP78, Collagen II, and β-actin were purchased from Proteintech (NJ, China). Horseradish peroxidase-labeled secondary antibodies, Alexa Fluor® 488-labeled goat anti-rabbit IgG (H + L) secondary antibody, and Alexa Fluor® 594-labeled goat anti-mouse IgG (H + L) secondary antibody were purchased from Abcam. Further, 4′,6-diamidino-2-phenylindole (DAPI) was purchased from Beyotime (SH, CHINA). The reagents for cell culture were obtained from Gibco (Grand Island, NY, United States).
4
Protein Expression Analysis of Distal Colon
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Distal colonic samples (2–3.5 cm proximal to the anus) were ground and lysed using a total protein extract kit (Keygen). Each sample (30 μg protein) was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Wang et al., 2016 (link)). The membrane was first incubated with primary antibodies against Caspase 3, PCNA, mTORC1, and GCN2 (Cell Signaling) overnight at 4°C and then incubated with the secondary antibody for 2 h at 25°C. The results were visualized via chemiluminescence with EZ-ECL (Biological Industries).
5
Molecular Signaling Profiling in mPFC Synapses
6
Cellular Signaling Pathway Analysis in Frozen Muscle
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Frozen gastrocnemius muscle tissue was ground into small pieces in liquid nitrogen and lysed in a lysis buffer containing cOmplete™ Protease Inhibitor Cocktail and PhosSTOP™ (Roche Diagnostics, Indianapolis, IN, USA), and then centrifuged. A Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA) was used to measure the protein concentration and same concentration of protein of each group was subjected to SDS-PAGE. After transferring, membranes were blocked by 5% skim milk and incubated with a primary antibody overnight. The primary antibodies used for Western blot were p-Akt, Akt, p-mTORc1, mTORc1, p-S6K1, S6K1, p-4E-BP1, 4E-BP1, p-FoxO3a, and FoxO3a (Cell signaling, MA, USA), MuRF1 and Atrogin-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and β-actin (GeneTex, Irvine, CA, USA). The next day, the membranes were incubated with the corresponding secondary antibodies for visualizing the protein bands using LAS3000 luminescent image analyzer (Fuji Film, Tokyo, Japan). β-actin was used as a loading control and Image J software (National Institute of Health, Bethesda, MD, USA) was used for quantification.
7
Immunoblot Analysis of Signaling Proteins
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U-87, SNB-19 and U-251 cells were lysed in 1% Nonidet P-40 lysis buffer 48 h after transfection with negative control ssRNA, miR-451, or miR-451 inhibitor oligonucleotide. Homogenates were clarified by centrifugation at 20,000 × g for 15 min at 4°C, and protein concentration was measured by the Lowry method. SDS-PAGE gels were loaded with 40 µg of protein from each sample, and resolved proteins were transferred to PVDF membranes (Millipore, Billerica, MA, USA) and incubated with primary antibodies against Rac1 (1:1,000 dilution; Abcam), AMPKα1, p-AMPKα1, mTORC1, p-mTORC1, confilin or p-confilin (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), followed by incubation with an HRP-conjugated secondary antibody (1:1,000 dilution; Zymed, San Diego, CA). The specific protein was detected using the SuperSignal protein detection kit (Pierce, Waltham, MA, USA). After washing with stripping buffer, the membrane was reprobed with antibody against GAPDH (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA).
8
Western Blot Quantification of Proteins
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Total proteins were quantified using the BCA assay kit (Beyotime), and equal amounts of protein (30 μg) were separated by 10% SDS-PAGE. After that, proteins were transferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% skim milk in tris-buffered saline with tween-20 (TBST) for 1 h, the membranes were incubated with primary antibodies: collagen 1α1 (1:1000, Cell Signaling Technology), fibronectin (1:1000, Abcam), alpha-smooth muscle actin (α-SMA; 1:1000, Abcam), TSC1 (1:1000, Abcam), phosphorylated-mTOR complex 1 (p-mTORC1; 1:1000, Cell Signaling Technology), mTORC1 (1:1000, Cell Signaling Technology), phosphorylated-p70 ribosomal s6 kinase (p-p70S6K; 1:1000, Cell Signaling Technology), p70S6K (1:1000, Cell Signaling Technology), phosphorylated-eukaryotic translation initiation factor 4E-binding protein 1 (p-4E-BP1; 1:1000, Cell Signaling Technology), 4E-BP1 (1:1000, Cell Signaling Technology), β-actin (1:1000, Abcam) at 4°C overnight. Later on, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h, and then the protein bands were visualized using the enhanced chemiluminescence (ECL) reagents (Thermo Fisher Scientific).
9
Genistein and Isoflurane Neuroprotective Mechanisms
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Genistein and isoflurane (0.75%) were obtained from Sigma-Aldrich (St. Louis, MO, USA). For expression analysis, antibodies against CREB, p-CREB, cleaved caspase-3, Bcl-2, Bad, Bcl-xL, Bax, β-actin, phosphatase and tensin homolog (PTEN), and mammalian target of rapamycin complex 1 (mTORc1) were purchased from Cell Signaling Technology (Beverly, MA, USA). Akt, p-Akt, GSK-3β, p- GSK-3β, BDNF, TrkB, and p-TrkB were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). PDE4 and Ca2+/calmodulin-dependent kinase IV (CaMKIV) were from Abcam. All other chemicals and reagents used in the present study were purchased from Sigma-Aldrich unless otherwise noted.
10
Molecular Signaling Pathway Analysis
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Primary antibodies against Bcl-2, Bax, Bad, and Bcl-xL were procured from Abcam (USA). Akt, p-Akt, mTORC1, p-mTORC1, mTORC2, p-mTORC2, phosphatase and tensin homolog (PTEN), glycogen synthase kinase 3β (GSK-3β), p- GSK-3β (Ser9), NF-κBp65, and cleaved caspase-3 were procured from Cell Signaling Technology (Danvers, MA, USA). CyclinD1, β-actin, and horseradish- and peroxidase-labeled IgG secondary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and used for expression analysis. Cell lysis buffer for Western blotting analysis was purchased from Beyotime Institute of Biotechnology (Beijing, China). Mangiferin and isoflurane were procured from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used for analysis were obtained from Sigma-Aldrich.
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