Human recombinant HGF was purchased from GenScript (Nanjing, China). The c-MET kinase inhibitors JNJ38877605 and rapamycin (RAPA) were obtained from Selleckchem (Houston, TX). Antibodies used include c-MET mAb, p-MET (Tyr1234/1235) mAb, Notch mAb, HES-1 mAb, and GAPDH mAb. Antibodies used in this study include c-MET mAb, p-MET (Tyr1234/1235) mAb (Abcam, U.S.A.), Notch mAb (Abcam, U.S.A.), HES-1 mAb (Abcam, U.S.A.), GAPDH mAb (Abcam, U.S.A.), Anti-mouse IgG (Abcam, U.S.A.), anti-CTLA4, #EPR1476 (Abcam, U.S.A.) and anti-human IgG4(Abcam, U.S.A.). All antibodies were purchased from Cell Signaling Technology, Inc.
Rapamycin rapa
Manufactured by Selleck Chemicals
Sourced in United States
Rapamycin (RAPA) is a macrolide compound used as a laboratory reagent. It functions as an immunosuppressant and has been studied for its potential therapeutic applications. Rapamycin acts by inhibiting the mammalian target of rapamycin (mTOR) signaling pathway, which is involved in cell growth, proliferation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
3 methyladenine 3 ma, by Selleck Chemicals (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Mhy1485, by Selleck Chemicals (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Jnj 38877605, by Selleck Chemicals (1 mentions)
Gapdh mab, by Abcam (1 mentions)
Anti human igg4, by Abcam (1 mentions)
3 methyladenine 3 ma, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti beclin 1, by Cell Signaling Technology (1 mentions)
Rabbit anti phospho ulk1, by Cell Signaling Technology (1 mentions)
Anti actin, by Abcam (1 mentions)
Pcdna3, by Addgene (1 mentions)
Prl tk, by Addgene (1 mentions)
Jagged1 fc, by R&D Systems (1 mentions)
Pgl3 basic, by Addgene (1 mentions)
Lenticrispr v2 plasmid, by Addgene (1 mentions)
Pvsv g, by Addgene (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Pspax2, by Addgene (1 mentions)
13 protocols using rapamycin rapa
1
HGF and c-MET Inhibitor Signaling Pathway Analysis
2
Autophagy Regulation by miR-34a
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Rabbit mAb to LC3I antibody (12741, dilution 1/800), rabbit mAb to p62 (23214, dilution 1/1000), rabbit anti-Beclin-1 (3495, diluted 1/1000), rabbit anti-Phospho-ULK1 (14202, diluted 1/800) and ULK1 (8054, diluted 1/1000) were all purchased from Cell Signaling Technology, Inc. Rabbit polyclonal anti-LC3II antibody (ab63817, dilution 1/1000) was purchased from Abcam. Anti-actin was obtained from Epitomics, an Abcam company (Cambridge, MA, USA). miR-34a inhibitors (5’-AGCCUUGCUGCAGGUGCGCAU-3’) and a nonsense control (NC) sequence (5’-UGCCUUACUGACGGUCGGAGA-3’) were obtained from Shanghai GeneChem, Inc. (Shanghai, China). According to the protocol, Huh7-R and HepG2-R cells (1 × 105) were transfected with 50 nM miR-34a inhibitors, 50 nM miR-34a mimics, 50 nM scramble control inhibitors or 50 nM scramble control mimics using Lipofectamine® 2000 (Thermo Fisher Scientific, Inc.) for ~ 30 min at room temperature. After 48 h of transfection, the transfected cells were used for subsequent experiments. 3-Methyladenine (3-MA), a class III phosphoinositol 3-kinase (PI3K) inhibitor, and choroquine (CQ), were used as selective inhibitors of autophagy, was purchased from Santa Cruz Biotechnology (Texas, U.S.A). Rapamycin (rapa), a potent and specific mTOR inhibitor used as an autophagy agonist, was obtained from Selleck.
3
Analyzing Cellular Signaling Pathways
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All information regarding antibodies used in this study is provided in Table S7 . Rapamycin (Rapa), everolimus (RAD001), deferoxamine (DFX), DAPT and MHY1485 were purchased from Selleck Chemicals (Houston, TX, USA). Jagged1-Fc was obtained from R&D system (Minneapolis, MN, USA). Lipofectamine RNAiMax was obtained from Invitrogen (Carlsbad, CA, USA). pRL-TK, pGL3-Basic, pcDNA3.0, pcDNA3.0-HA-HIF-1α, lenti-CRISPRv2 plasmids and packaging vectors (pVSVG and psPAX2) were purchased from Addgene (Cambridge, MA, USA).
4
Chondrocyte Inflammatory and Catabolic Regulation
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The list of regents was obtained commercially: Recombinant Rat interleukin 1 beta (IL-1β) (501-RL-010) was acquired from R&D Systems (Minneapolis, MN, USA). Rapamycin (Rapa) (S1039) was acquired from Selleck (Houston, USA) and diluted in DMSO. Antibodies against COX2 (12882; 1:1000), YAP (14074; 1:1000), P-P65 (3033; 1:1000), P-YAP (13008; 1:1000) and P65 (8242; 1:1000) were gained from Cell Signaling Technology Inc. (Beverly, USA). Antibodies against MMP13 (18165-1-AP; 1:1000), COL2A1 (28459-1-AP; 1:800), GAPDH (10494-1-AP; 1:5000), P-RIPK1 (66854-1-Ig; 1:2000) and β-actin (CL594-66009; 1:5000) were acquired from Proteintech Group (Wuhan, China). Antibodies against iNOS (A0312; 1:1000) and aggrecan (A11691; 1:1000) were acquired from Abclonal (Wuhan, China). Antibodies against P62 (GB11531-100; 1:1000) was acquired from Servicebio (Wuhan, China). Antibodies against MMP3 (BM4074; 1:500), FITC-conjugate goat anti-mouse and anti-rabbit secondary antibodies, type II collagenase and trypsin were purchased from Boster (Wuhan, China). IP lysis buffer (p0013) was provided by Beyotime (Shanghai, China).
5
Kae Regulates Osteogenic Differentiation
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Kae (purity>98%) was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Kae was dissolved in DMSO and diluted to 0.01% in PBS. Rapamycin (Rapa), a specific inhibitor of mTOR, was purchased from Selleck Chemicals. The Alizarin Red S (ARS) staining buffer and alkaline phosphatase (ALP) detection kits were purchased from Nanjing Jiancheng Bioengineering Institute. Anti-runt-related transcription factor 2 (Runx2; cat. no. ab23981) and anti-Osterix (cat. no. ab22552) was purchased from Abcam. Anti-eukaryotic translation initiation factor 4E-binding protein 1 (4E/BP1; cat. no. 94525), anti-phosphorylated (p)-4E/BP 1 (cat. no. 2855) and anti-ribosomal protein S6 kinase B1 (S6K1; cat. no. 9202), anti-p-S6K1 (cat. no. 9204) was obtained from Cell Signaling Technologies, Inc. Horseradish peroxidase-labeled anti-immunoglobulin G secondary antibody (goat anti-mouse lgG; cat. no. SA00001-1; and goat anti-rabbit lgG; cat. no. SA00001-2) was obtained from ProteinTech Group, Inc. Anti-β-actin antibody (cat. no. KL002) was provided by Nanjing Jiancheng Bioengineering Institute.
6
Honokiol and Rapamycin Signaling Pathways
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Honokiol (HNK) and Rapamycin (RAPA) were purchased from Selleckchem. For Western blot analysis, primary antibodies against CDK2, CDK4, CDK6, Cyclin D1, p21, Bcl-2, Bcl-xL, c-Met, phospho-c-Met, HO-1, Akt, mTOR and S6 and species-specific secondary antibodies were obtained from Cell Signaling. Antibody against β-actin was purchased from Sigma Aldrich. Anti-PD-L1, phospho-Akt, phospho-S6 and phospho-mTOR antibodies were purchased from Thermo Fisher Scientific. Recombinant human hepatocyte growth factor (HGF) was purchased from Peprotech.
7
Neuroprotective Role of MEG3 in OGD/R
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Si-MEG3, si-NC (control), miR-181c-5p mimic, NC mimic, miR-181c-5p inhibitor, NC inhibitor, pcDNA-3.1, and pcDNA-3.1-MEG3 were synthesized by GenePharma (Shanghai, China). Rapamycin (RAPA; a mammalian target of rapamycin inhibitor) was purchased from Selleck (Cat# 53123-88-9, Houston, TX, USA). HT22 cells were seeded in 24-well plates; when they reached more than 80% confluence, they were transfected with pcDNA3.1/si-NC (100 μM) or co-transfected with MEG3-WT/MEG3-MUT (100 μM) and miR-181c-5p mimic/NC mimic (100 μM) for 48 hours. Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) was used as the transfection reagent according to standard protocols. RAPA (200 nM) was added to the cells and cultured for 48 hours. After transfection or incubation with RAPA, cells were harvested for further analysis. This procedure was performed before OGD/reoxygenation (OGD/R) treatment.
8
Oxidative Stress Impact on hiPSC-CMs
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hiPSC-CMs were intermittently exposed Lipopolysaccharide (LPS) (1 ug/ml, Sigma, St. Louis, MO,US) every 6 hours per day for 1 week, observing the long-term effects of oxidative stress on WT or PLEKHM2-KO hiPSC-CMs at day 40 post myocardial differentiation. The effect of inhibiting oxidative stress on PLEKHM2-KO hiPSC-CMs was examined by treating with glutathione (GSH) (2 mM, Sangon Biotech, Shanghai, China) for 10 days, and the culture medium with GSH was changed daily until day 40 post myocardial differentiation. Rapamycin (RAPA) (500 nM, Selleck Chemicals, Houston, TX, US) was used to inhibit the activation of the mTORC1 signaling pathway for 72 hours, and evaluating the effects of RAPA on autophagic flux, mitochondrial function, and myocardial contractility in WT or PLEKHM2-KO hiPSCs-CMS at D40 post myocardial differentiation.
9
Establishing Primary Neuronal Cultures
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Primary neuronal cultures were established as previously described (Guo et al., 2020 (link)). Postnatal brain tissue was dissected from early postnatal mice, and the tissues were digested with trypsin and mechanical dissociation to obtain neurons. The cell suspension was diluted with DMEM supplemented with 20% FBS (Gibco, Thermo Fisher Scientific). Neurons were plated at a density of 100,000 cells on poly-L-lysine–coated 35-mm dishes or glass coverslips in 6-well plates and incubated in a cell culture incubator at 37°C for 4 h. Four hours after plating, the cells were maintained in Neurobasal medium supplemented with B27, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Neurons were transfected with a plasmid encoding green fluorescent protein (GFP) using the calcium phosphate transfection method at 7 days (DIV7) in vitro to visualize their spines. Certain cells were treated with 10 μM 3-methyladenine (3-MA) or 100 nM rapamycin (RAPA) (Selleck, United States) to assess the effects of drug interventions.
10
Investigating Autophagy Regulation Mechanisms
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Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco/Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco's phosphate-buffered saline (DPBS) was purchased from Invitrogen/Thermo Fisher Scientific. In addition, 3-methyladenine (3-MA) and rapamycin (RAPA) were purchased from Selleck Chemicals (Shanghai, China), and leptomycin B (LMB) was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). The anti-Nur77 antibody was purchased from Novus Biologicals, and anti-LC3 (cat. nos. 12741 and 2775) and anti-GAPDH antibodies (cat. no. 5174) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Beclin-1 (cat. no. ab210498) and anti-Bcl2 (cat. no. ab692) antibodies were purchased from Abcam (Cambridge, MA, USA). Anti-rabbit and mouse IgG (ZB2301, ZB2305, 1:2,000) were purchased from Zhong Shan Golden Bridge Biotechnology Co., Ltd. (Beijing, China).
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