Proteins (30 μg) were resolved on a 4–12% Bis–Tris gradient SDS–PAGE under reducing conditions and transferred onto nitrocellulose membrane. The primary antibodies were RANKL, E-cadherin, vimentin, OPG, c-Met (Santa Cruz Biotechnology, Inc.), p-c-Met (Tyr-1230/34/35; Invitrogen), RANK (Amgen, Thousand Oaks, CA, USA), and N-cadherin (BD Transduction Laboratories, San Jose, CA, USA). AR (441), Chr-A (H-300), synaptophysin (SYP; D4), CD44 (DF1485), Sox-2 (Y-17), Nanog (5A10), LIN-28 (H-44) (Santa Cruz Biotechnology, Inc.), FOXA2 (D56D6; Cell Signaling Technology, Danvers, MA, USA), and PROM1 (CD133; Abnova, Taipei City, Taiwan) antibodies were used to detect neuroendocrine and stem cell differentiation. c-Myc (D84C12XP), Lamin A/C (Cell Signaling Technology), and Max (sc-197X) antibodies (Santa Cruz Biotechnology, Inc.) were used for nuclear protein detection.
C met
Manufactured by Santa Cruz Biotechnology
Sourced in United States
C-Met is a protein that plays a crucial role in cellular signaling pathways. It functions as a receptor tyrosine kinase, which means it can bind to and activate other proteins involved in cell growth, survival, and motility. C-Met is an important target for research in various fields, including cancer biology and developmental biology.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Santa Cruz Biotechnology (3 mentions)
Gapdh, by Santa Cruz Biotechnology (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
P akt, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ecl kit, by Merck Group (2 mentions)
Gapdh antibody, by Santa Cruz Biotechnology (2 mentions)
N cadherin, by Santa Cruz Biotechnology (2 mentions)
N cadherin, by BD (1 mentions)
Foxa2 d56d6, by Cell Signaling Technology (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Rankl, by Santa Cruz Biotechnology (1 mentions)
P c met, by Thermo Fisher Scientific (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Clarity western ecl, by Bio-Rad (1 mentions)
P met, by Cell Signaling Technology (1 mentions)
P4ebp, by Cell Signaling Technology (1 mentions)
P src, by GeneTex (1 mentions)
20 protocols using c met
1
Western Blot Analysis of Protein Expression
2
Western Blot Assay for Protein Analysis
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Western blot assays were performed using the following antibodies: MCL-1 MoAb used at a dilution of 1:1000 (4572 Cell Signaling), vinculin MoAb (Sigma-Aldrich, dilution of 1:10,000), C-MET (Santa Cruz, 1:1000), P-MET (Cell Signaling, 1:1000), 4EBP (Cell Signaling, 1:1000), P-4EBP (Cell Signalling, 1:1000), S6 (Cell Signaling, 1:1000) and P-S6 (Cell Signaling, 1:1000). Antibodies were purchased from the indicated sources.
Cells were seeded (1.5 × 106 in adhesion and 4 × 106 in suspension) and resuspended after drug treatment in a urea buffer; then protein concentration was determined by a protein assay kit (Bio-Rad). Proteins (80 µg) were separated in 12% SDS–polyacrylamide gels, transferred onto a nitrocellulose membrane and probed against MCL-1 MoAb. The secondary antibody was mouse IgGκ-binding protein HRP. Proteins were visualized with Clarity Western ECL and analyzed with Chemidoc (Bio-Rad).
Cells were seeded (1.5 × 106 in adhesion and 4 × 106 in suspension) and resuspended after drug treatment in a urea buffer; then protein concentration was determined by a protein assay kit (Bio-Rad). Proteins (80 µg) were separated in 12% SDS–polyacrylamide gels, transferred onto a nitrocellulose membrane and probed against MCL-1 MoAb. The secondary antibody was mouse IgGκ-binding protein HRP. Proteins were visualized with Clarity Western ECL and analyzed with Chemidoc (Bio-Rad).
3
Antibody and Kinase Inhibitor Sources
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Rabbit polyclonal antibodies of Hic-5, p-Src, p-AKT, and AKT were purchased from GeneTex (Irvine, CA, USA) whereas mouse monoclonal antibodies of EGFR, c-Met, Her2, Her3, p-JNK, JNK and rabbit polyclonal antibody of c-Src were from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Src kinase inhibitors PP1 were from Merck (Darmstadt, Germany) and dasatinib was from Tocris Bioscience (Bristol, UK).
4
Regulation of Osteoblast Differentiation
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Recombinant mouse vaspin was purchased from the Enzo Life Sciences Inc. (Farmingdale, NY, USA). Antibodies for Runx2, β-actin, Akt, p-Akt, and c-met were purchased from Santa Cruz Biotechnology Inc. (Waltham, MA, USA). Akt inhibitor LY294002 was purchased from Calbiochem Corp. (San Diego, CA, USA). MiR-34c mimics and miR-34c inhibitors and their control oligos were purchased from Ribobio Co., Ltd (Guangzhou, China). The growth medium and foetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco-BRL Co., Ltd (Grand Island, NY, USA). Ascorbic acid and β-glycerophosphate (β-GP), and Alizarin red S were purchased from Sigma Chemical Co., Ltd (St.Louis, MO, USA). All the experimental procedures were approved by the Ethics Committee of the Second Xiangya Hospital of Central South University, China and carried out in accordance with the approved guidelines.
5
Western Blot Analysis of c-Met in HepG2 and SMMC7721 Cells
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HepG2 and SMMC7721 cells were solubilized in RIPA lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.) to extract total protein. After boiling for 5–10 min, the protein samples extracted from different cell suspensions were separated by SDS-PAGE and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk prepared in Tris-buffer and incubated with the primary antibodies directed against c-met (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.) for 1 h at room temperature. The membrane was then incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Santa Cruz Biotechnology, Inc.), for 4 h at room temperature. The blot was developed using The ECL™ Western Blotting Detection Reagent (GE Healthcare) and the proteins were visualized by enhanced chemiluminescence (Amersham Biosciences).
6
Immunofluorescence Analysis of GBM Markers
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Human GBM and normal brain tissues were obtained from the Cooperative Human Tissue Network (CHTN). Anti-FRMD6 (Sigma), -Lats1/2 (Bethyl Lab), -MST1/2, -YAP, -c-Met, -PDGFRA, -PDGFRB, and -merlin (Santa Cruz), -actin (Sigma), -v5 epitope (Invitrogen), -phospho-Lats1, -phospho-YAP (Cell signaling), -phospho-c-Met (Invitrogen and Santa Cruz), and -phospho-PDGFRA/B (Santa Cruz and R & D Systems) antibodies were used in the experiments. Anti-Ki67 was from the Fisher Scientific. Secondary anti-rabbit Alexa Fluor® 594 and anti-mouse Alexa Fluor® 488 were from Invitrogen.
7
Immunoblotting Antibody Validation Protocol
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Primary antibodies used for immunoblotting were as follows: total and cleaved forms of PARP and caspase-3, phospho-KIT Y719, phospho-AKT S473, AKT, ABCG2, phospho-FGFR Y653/654 (Cell Signaling, Danvers, MA, USA), FAK, phospho-FAK Y397 (BD Biosciences, Franklin Lakes, NJ, USA), KIT (DakoCytomation), phospho-Met Tyr1230/1234/1235 (R&D Systems, Minneapolis, MN, USA), Axl, FGFR2 (Sigma), c-Met, MDR, MRP-1 and actin (Santa Cruz Biotechnology, Dallas, TX, USA). The HRP-conjugated secondary antibodies for Western blotting were purchased from Santa Cruz Biotechnology.
8
Antibody Detection for Protein Analysis
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Antibodies against ETS-1, P53, MMP1, MMP9, u-PA, c-Met, and GAPDH were from Santa Cruz Biotechnology, USA and antibodies against FBI-1 from Sigma Technology, St. Louis, USA. A polyclonal anti-rabbit IgG antibody and monoclonal anti-Flag monoclonal antibody both conjugated with the HRP (horseradish peroxidase, HRP) were from Sigma, St. Louis, USA.
9
Investigating Apoptosis Mechanisms in Cancer
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Roswell Park Memorial Institute-1640 (RPMI-1640; Booster Biological Technology Co, Ltd, Wuhan, People’s Republic of China), fetal bovine serum (Biological Industries, Beit Haemek, Israel), trypsin & 0.02% EDTA (Gino Biopharmaceutical Technology, Hangzhou, People’s Republic of China), MMC (TCI [Shanghai] Development Co., Ltd., Shanghai, People’s Republic of China), CTX (Abcam, Cambridge, UK), DDP (HaoSen, Jiangsu, People’s Republic of China), and Cell Counting Kit 8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan), bovine serum albumin (Amresco, Solon, OH, USA) were bought and utilized. HER-2, c-Met, p53, p27, p21, TERT, Pol α, Pol ε, Bcl xl, Bax, β-Actin, GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA), c-Kit, NF-κB-p65, Akt, p44/42 MAPK, PCNA, PARP, caspase 9, pro-caspase 3, cleaved caspase 3 (Cell Signaling Technology, Danvers, MA, USA), and Bcl 2 (Abcam) were used. Secondary antibodies used were anti-goat, anti-rabbit, and anti-mouse (Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd, Beijing, People’s Republic of China). HRP substrate (Millipore Corporation, Billerica, MA, USA), skim-milk (BD-Difco, Sparks, MD, USA), MitoTracker® Red CMXRos (Invitrogen Life Technologies Corporation, Carlsbad, CA, USA), PI/RNase Staining Buffer (BD Biosciences, San Diego, CA, USA), and PE-Annexin-V apoptosis detection kit (BD Biosciences) were also purchased and used.
10
Immunohistochemical Analysis of Gastric Cancer Biomarkers
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Consecutive 4-μm sections were immunohistochemically stained using the immunoperoxidase technique described previously [38 (link)], with primary antibodies against c-Met (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), HMGB1 (Proteintech Group, Rosemont, IL, USA), RegIV (Biorbyt, St. Louis, MO, USA), and PCDHB8 established in our laboratory [13 (link)], and appropriate secondary antibodies (Medical and Biological Laboratories [39 (link)], Nagoya, Japan) (all 0.2 µg/mL). The tissue sections were then color-developed with diamine benzidine hydrochloride (DAKO, Glostrup, Denmark) and counterstained with Meyer’s hematoxylin (Sigma). For assessing the expression of c-Met, RegIV, and PCDHB9, cells that exhibited immunoreactivity at the cytoplasmic membrane were counted, and the staining intensity was scored between 0 to 1, (where a score of 0.3 was used to describe the expression level in a normal gastric foveolar epithelium). The staining positivity (0–100) was then calculated as the staining strength score multiplied by the staining area (%). The expression of HMGB1 was assessed by determining the percentage of nuclear immunoreactivity in 1000 examined nuclei.
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