For transient transfection of hippocampal neurons, 300 ng of plasmid DNA were transfected using 1 μl of Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) in 100 μl of the plating medium. Neurons were transfected at DIV 8 and were fixed after two additional days of incubation. Plasmids used were as follows: pEGFP-N1 (Takara Bio, Mountain View, CA, USA), mCherry-Rab5CA (Q79L; Addgene, #35138), mCherry-Rab5DN (S34N; Addgene, #35139), mRFP-Rab5 (Addgene, #14437), GFP-rab7 WT (Addgene, #12605), GFP-rab7 DN (Addgene, #12660), EGFP-Rab7A Q67L (Addgene, #28049), EGFP-Rab4A (Addgene, #49434), EGFP-Rab4AQ67L (Addgene, #49475), EGFP-Rab4AS22N (Addgene, #49476), HA-Rab11-DN (S25N), (Addgene, #101046), HA-Rab11-WT (Addgene, #101047), EGFP-Rab11AQ70L (Addgene, #49553), HA-Rab8a-WT (Addgene, #101048), HA-Rab8a-DN (T22N; Addgene, #101049), and HA-Rab8a-CA (Q67L) (Addgene, #101050). Inhibitors were dissolved in dimethylsulfoxide (DMSO), HGF (Merck KGaA, Darmstadt, Germany) in PBS and added to hippocampal neurons (DIV 9) to final concentrations of 50 ng/ml for HGF, 200 nm for mTOR inhibitor rapamycin (Merck), and 1 μM PHA-665752 (Merck KGaA, Darmstadt, Germany). Analyses were performed 30 min and 24 h (HGF Stimulation) or 24 h (Rapamycin treatment) after the addition of the reagents.
Pha 665752
Manufactured by Merck Group
Sourced in United States, Germany
PHA-665752 is a laboratory reagent produced by Merck Group. It is a small molecule compound that functions as a selective inhibitor. Further details on its specific applications or intended uses are not available.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Anti stat3, by Cell Signaling Technology (2 mentions)
Anti actin, by Santa Cruz Biotechnology (2 mentions)
Tgf β1, by Sino Biological (2 mentions)
Mcherry rab5ca, by Addgene (1 mentions)
Mcherry rab5dn, by Addgene (1 mentions)
Egfp rab4aq67l, by Addgene (1 mentions)
Egfp rab4as22n, by Addgene (1 mentions)
Egfp rab11aq70l, by Addgene (1 mentions)
Mtor inhibitor rapamycin, by Merck Group (1 mentions)
Hepatocyte growth factor (hgf), by Merck Group (1 mentions)
Pegfp n1, by Takara Bio (1 mentions)
Mrfp rab5, by Addgene (1 mentions)
Gfp rab7 wt, by Addgene (1 mentions)
Egfp rab7a q67l, by Addgene (1 mentions)
Egfp rab4a, by Addgene (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Anti c met, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
16 protocols using pha 665752
1
Transient Transfection of Hippocampal Neurons
2
Monoclonal Antibody and Kinase Inhibitor Assay
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Anti-human-IDO monoclonal antibody was prepared and utilized as previously reported (8 (link)). Anti-human-actin(Sigma), anti-mouse-CD49b(R&DSystems), anti-HGF-α (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-phospho-c-Met, anti-c-Met, anti-phospho-AKT, anti-AKT, anti-phospho-ERK, anti-ERK, anti-phospho-STAT3 and anti-STAT3 (Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies were purchased and utilized according to the manufacturer's instructions.
The c-Met tyrosine kinase inhibitor PHA-665752((3Z)-5-[(2,6-dichlorobenzyl)sulfonyl]-3-[(3,5-dimethyl-4-{[(2R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl]carbonyl}-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one; Merck KGaA, Darmstadt, Germany) (28 (link)), the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; Cell Signaling Technology) (29 (link)), the MEK1/2 inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene; Cell Signaling Technology) (30 (link)), and the STAT3 inhibitor WP1066 ((2E)-3-(6-Bromo-2-pyridinyl)-2-cyano-N-[(1S)-1-phenylethy]-2-propenamide P; Santa Cruz Biotechnology) (31 (link)) were purchased and were utilized according to the corresponding manufacturer's instructions.
The c-Met tyrosine kinase inhibitor PHA-665752((3Z)-5-[(2,6-dichlorobenzyl)sulfonyl]-3-[(3,5-dimethyl-4-{[(2R)-2-(pyrrolidin-1-ylmethyl)pyrrolidin-1-yl]carbonyl}-1H-pyrrol-2-yl)methylene]-1,3-dihydro-2H-indol-2-one; Merck KGaA, Darmstadt, Germany) (28 (link)), the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; Cell Signaling Technology) (29 (link)), the MEK1/2 inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene; Cell Signaling Technology) (30 (link)), and the STAT3 inhibitor WP1066 ((2E)-3-(6-Bromo-2-pyridinyl)-2-cyano-N-[(1S)-1-phenylethy]-2-propenamide P; Santa Cruz Biotechnology) (31 (link)) were purchased and were utilized according to the corresponding manufacturer's instructions.
3
Lung and Gastric Cancer Cell Lines for MET Inhibitor Evaluation
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The lung adenocarcinoma cell lines H596 (harboring the MET exon 14 skipping mutation) and H1993 (MET amplification), the gastric carcinoma cell line Hs746T (both the MET exon 14 skipping mutation and amplification), and the non-neoplastic human bronchial epithelial cell line BEAS-2B were used in this study. H596 and Hs746T cells were kindly provided by James G. Christensen (Mirati Therapeutics, San Diego, CA, USA), and H1993 cells by Sukjoon Yoon (Sookmyung Women’s University, Seoul, Korea). BEAS-2B (ATCC CRL-9609) cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Hs746T cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics, while H596 and H1993 cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% antibiotics, and BEAS-2B cells were in DMEM/F12 (1:1) with 5% FBS and 1% antibiotics. All cell lines were kept in a humidified atmosphere of 5% CO2 at 37 °C. PHA665752 (a MET inhibitor) was purchased from Sigma-Aldrich (St. Louis, MO, USA), crizotinib came from Selleckchem (Houston, TX, USA), and rhHGF was from ProSpec-Tany TechnoGene Ltd. (Ness Ziona, Israel).
4
Exploring Renal Distal Tubule Epithelium Signaling
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A canine renal distal tubular epithelium cell line (MDCK) was purchased from the American Type Culture Collection (Rockville, USA). RSG, GW9662, and PHA665752 were obtained from Sigma-Aldrich (St. Louis, MO, USA). The following primary antibodies were used: p-Met, Smad7, TGF-β1, and PPAR-γ (Santa Cruz, USA); HGF (Abcam, USA); p-PPAR-γ (Ser112) (Bioss, China); c-Met (Proteintech, China); Smad2, Smad3, p-Smad2 3, and Lamin B (Wanleibio, China); and GAPDH (Zhongshan Golden Bridge Bio Co., Ltd., China). The secondary antibodies, including HRP-conjugated AffiniPure goat anti-rabbit IgG and HRP-conjugated AffiniPure goat anti-mouse IgG, were purchased from Zhongshan Golden Bridge Bio Co., Ltd. (Beijing, China). A Nuclear and Cytoplasmic Protein Extraction Kit was acquired from Beyotime Institute of Biotechnology (Shanghai, China).
5
Cell Viability Assay with Inhibitors
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Cell growth over time was measured using the PrestoBlue Cell Viability Reagent (Invitrogen). Briefly, 67NR, 5637, SW480 or MDA-468 cells were seeded in 96-well plates (2,500 cells/well) and exposed to ATX-101 (25 amino acid cell-penetrating APIM-containing peptide [21 (link)], Ac-MD-RWLVK-W-KKKRK-I-RRRRRRRRRRR, Invitrogen, Sweden) (4-12 μM), ATX-A (Ac-MD-RALVK-W-KKKRK-I-RRRRRRRRRRR, Invitrogen, Sweden) (6 μM), AEE788 (EGFR/HER2/VEGFR inhibitor, Selleck Chem) (0.5-1 μM), Bafilomycin A1 (10 nM) Cell Signaling, #54645, or cMet inhibitor (PHA-665752, Sigma, 2 μM) until harvest at day three. Data displayed are percentage viability relative to untreated control in one representative experiment out of three biological replicas demonstrating the same trend.
6
Inhibition of MET, EGFR, and PI3K Pathways
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The selective ATP competitive MET inhibitor PHA665752 was purchased from Sigma-Aldrich, and the EGFRs inhibitor lapatinib, the PI3K inhibitor LY294002 and the STAT3 inhibitor LLL12 were purchased from Selleckchem. The cabozantinib and crizotinib RTKs inhibitors were kindly provided by B. Blanchet (Cochin Hospital, Paris). The inhibitors were diluted in dimethylsulfoxide (DMSO).
The following antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): anti-MET (sc-10), anti-P-ERK1/2 (sc-7383), and anti-GAPDH. The anti-P-MET (Tyr1234/1235)(D26), anti-ERK 1/2, anti-P-AKT (Ser473), anti-P-AKT (Thr308), anti-AKT, anti-P-STAT3 (D3A7), anti-STAT3, anti-P-EGFR (Tyr1068), anti-EGFR antibodies and anti-cleaved caspase-3 (Asp15) were from Cell Signaling Technology, and the anti-actin antibody was from Sigma-Aldrich.
The following antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): anti-MET (sc-10), anti-P-ERK1/2 (sc-7383), and anti-GAPDH. The anti-P-MET (Tyr1234/1235)(D26), anti-ERK 1/2, anti-P-AKT (Ser473), anti-P-AKT (Thr308), anti-AKT, anti-P-STAT3 (D3A7), anti-STAT3, anti-P-EGFR (Tyr1068), anti-EGFR antibodies and anti-cleaved caspase-3 (Asp15) were from Cell Signaling Technology, and the anti-actin antibody was from Sigma-Aldrich.
7
Cell Viability Assay with ADCs
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Cells diluted in 10% FBS containing growth media were plated in 96-well flat-bottom plates (Corning, Inc., Corning, NY, USA.) and incubated with the indicated ADCs, controls, or c-Met inhibitor PHA-665752 (Sigma Aldrich) for 5 days. Cell viability was measured by detecting ATP with Celltiter-Glo reagent (Promega, Madison, WI) on a Perkin Elmer EnSpire multimode plate reader (Perkin Elmer, Waltham, MA). Half-maximal inhibitory concentrations (IC50) were calculated by non-linear regression analysis using a sigmoidal curve fitting with Prism 7 software (GraphPad, La Jolla, CA).
8
Colon Cancer Cell Line Characterization
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Colon cancer SW480, DLD-1, HCT-116, HT-29, RKO and Caco-2 cells were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). DiFi cells were purchased from Shanghai Yan Cheng biological technology Co., Ltd. (Shanghai, China). SW480 cells were grown in Leibovitz’s L-15 medium (Gibco, Gaithersburg, MD, USA), DiFi cells were grown in McCoy’s 5A medium (Gibco, Gaithersburg, MD, USA), and all the other cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Gaithersburg, MD, USA). Media were supplemented with 10% fetal bovine serum (FBS). Cetuximab was obtained from Merck KgaA (Darmstadt, Germany). PHA-665752 and PP2 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dasatinib was purchased from Selleck chemicals (Houston, TX, USA). Antibodies to MET, phospho-MET (Tyr1234/1235), EGFR, phospho-EGFR (Tyr1068), SRC, phospho-SRC (Y416), poly (ADP-ribose) polymerase (PARP), AKT, phospho-AKT (Ser473), phospho-ERK1/ERK2 (Thr202/Tyr204), Caspase-3 were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-Actin, anti-ERK, secondary goat anti-rabbit and goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
9
TGF-β1 and PHA665752 Modulate Fibronectin Expression
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MRC-5 cells were plated at a density of 2 × 105 cells/well in 6-well culture plates (BD Biosciences) and cells were treated with or without indicated concentrations of PHA665752 (Sigma-Aldrich) and 2.5 ng/mL TGF-β1 (Sino Biological) for 24 hours. After removing the medium, hypoxia-pretreated MSCs in transwells were co-cultured with the PHA665752-treated MRC-5 cells for 24 hours. The MRC-5 cells were harvested for detection of fibronectin mRNA expression level by quantitative real-time RT-PCR.
10
MTT Assay for Cell Viability
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Cells were plated in 100 μl complete medium in 96-well flat bottom plates in the presence or absence of crizotinib (500 nM) or vehicle (DMSO) or increasing concentrations of PHA665752 (Sigma) and Savolitinib (Selleckchem). After 24 h (crizotinib) or 72 h (PHA665752, Savolitinib) of incubation 10 μl of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution dissolved in PBS was added for 2 h followed by the addition of 100 μl of solubilization solution (40% dimethylformamide, 2% acetic acid, 16% sodium dodecyl sulfate, pH 4.7) and rigorous shaking to dissolve the formazan crystals. The absorbance at 570 nm was determined using a Tecan plate reader (Tecan, Austria). Triplicate wells were assayed for each condition.
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