The nucleus and cytoplasmic fractions of CCA cells were prepared as previously described25 (link). Cell lysates of the entire cells or nucleus fractions were collected by digesting with RIPA buffer and protease inhibitors (Sigma-Aldrich, USA). Protein concentration was determined by BCA kit (Thermo Fisher, USA). A total of 30 μg protein was loaded on 10% or 15% SDS-PAGE gels and transferred onto nitrocellulose membranes. The membranes were incubated with specific first antibodies and corresponding second antibody. The specific antibodies were listed below: Cleaved Caspase-3 #9664, PCNA #13110; MMP-3 #14351, ZEB1 #70512, E-Cadherin #3195, Vimentin #5741, Oct-4 #2750, LIN28A #3695, Nanog #3580, Sox2 #3579, β-Catenin #8480, Phospho-β-Catenin (Ser33/37/Thr41) # 9561, Phospho-GSK-3β (Ser9) #5558, GSK-3β#12456, c-Myc #9402, Survivin #2803, Lamin B1 #12586, and GAPDH #5174 were purchased from Cell Signaling Technology with a work concentration at 1: 1000. BCL9L #ab233736 was purchased from Abcam with a work concentration at 1: 500. The second antibodies were goat anti-rabbit IgG HRP-linked antibody (Cell signaling #7074; 1:4000) and sheep anti-mouse IgG-HRP (GE/Amershan #NXA931; 1: 5000).
C myc 9402
Manufactured by Cell Signaling Technology
Sourced in United States
c-Myc (9402) is an antibody product from Cell Signaling Technology. It is a rabbit monoclonal antibody that recognizes the c-Myc protein.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Phospho gsk 3β ser9, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3 9664, by Cell Signaling Technology (1 mentions)
Gsk 3β 12456, by Cell Signaling Technology (1 mentions)
Lamin b1 12586, by Cell Signaling Technology (1 mentions)
Lin28a, by Cell Signaling Technology (1 mentions)
Phospho β catenin ser33 37 thr41, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Cell Signaling Technology (1 mentions)
Mmp 3, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Sirt1 2310, by Cell Signaling Technology (1 mentions)
Irdye 680 goat anti mouse or anti rabbit igg, by LI COR (1 mentions)
5 protocols using c myc 9402
1
Immunoblotting Analysis of Cellular Fractions
2
Synthesis and Characterization of Histone Deacetylase Inhibitors
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AN446 was synthesized as described [10 ]; HDAC inhibitors—SAHA and entinostat (MS-275)—were obtained from Cayman Chemical (Ann Arbor, MI, USA), romidepsin (depsipeptide) from ApexBio Tech LLC (Houston, TX, USA), and panobinostat (LBH589) and belinostat (PXD101) from MedChem Express (Princeton, NJ, USA). These HDACIs were dissolved in DMSO to a concentration of 100 mM and stored in aliquots at −20 °C. Dox hydrochloride 2 mg/mL, was obtained from Ebewe Pharma Ges.m.b.H. (Unterach, Austria) and was diluted in saline solution. Polyclonal antibodies: c-Myc #9402, SIRT1 #2310 (Cell Signaling, Danvers, MA, USA) and superoxide dismutase 1(SOD1)#ab13498 (Abcam, Cambridge, UK), phospho-H2AX, Serine 139, pH2AX # A300-081A-M (Bethyl Laboratories, Montgomery, TX, USA), cyclooxygenase-2 (COX-2) #PA5-16817 (ThermoFisher, Rockford, IL, USA), Ki-67, BRB040 (Zytomed, Berlin, Germany). Mouse monoclonal antibodies: Rad51 #05-530-I (Sigma, Saint Louis, MO, USA), and actin # SKU:0869100-CF (MP Biomedicals, Aurora, OH, USA). Secondary antibodies: IRDye® 680 goat anti-mouse or anti-rabbit IgG (LI-COR Biosciences, Lincoln, NE, USA), and biotinylated goat anti-rabbit IgG-B (Santa Cruz Biotechnology, CA, USA).
3
Protein Extraction and Western Blotting
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Total protein was lysed by using lysis buffer containing 50 mM Tris (pH7.5), 150 mM NaCl, 10% glycerol, 0.5% Nonidet P-40 (NP-40), with addition of protease and phosphatase inhibitors (1:100). The lysates were subjected to SDS-PAGE under standard conditions. The following antibodies were used: rabbit polyclonal WWOX 1:10,000, mouse monoclonal Gapdh (CB-1001-), Calbiochem 1:10,000, mouse monoclonal p21(F-5), sc 6246, Santa Cruz 1:200, mouse monoclonal p53 (1C12), Cell Signaling, 1:1000, mouse monoclonal MCM7 (141.2), sc-9966- Santa Cruz 1:200, rabbit polyclonal c-Myc (9402)-Cell Signaling, 1:750, rabbit polyclonal DsRed (632496), TaKaRa, 1:500, and mouse monoclonal β-actin (A5441), Sigma 1:10,000.
4
Quantification of YAP, TEAD1, and c-Myc Protein Expression
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Cells were seeded in 6-well plates at a density of 800,000 cells per well. The following day, culture medium was replaced by new medium with the addition of 1.25 ug/mL VP, 7.5 ug/mL VP or PBS. After 24-hour incubation, cells were washed with ice-cold PBS and lysed with MPER with a protease and phosphatase inhibitor. Samples were sonicated for 15 seconds, and centrifugated for 20 minutes at 14,000 g in a precooled 4°C centrifuge. The supernatant was used for further analyses.
Per lane, 20 ug of protein were loaded on a 4% to 12% Bis-Tris gel (NuPage, Invitrogen). After electrophoresis, the assay was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Coomassie blue staining was used to ensure equal loading. The membrane was blocked for 1 hour at room temperature in 5% milk and incubated for 3 hours with the respective primary antibodies at a 1:1000 dilution. After washing, the membrane was incubated for 1.5 hours with the respective secondary antibodies at a 1:2000 dilution. Protein expression was visualized with the ECL technique (Amersham ECL Select). Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): YAP (4912S), TEAD1 (12292S), and c-Myc (9402S).
Per lane, 20 ug of protein were loaded on a 4% to 12% Bis-Tris gel (NuPage, Invitrogen). After electrophoresis, the assay was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). Coomassie blue staining was used to ensure equal loading. The membrane was blocked for 1 hour at room temperature in 5% milk and incubated for 3 hours with the respective primary antibodies at a 1:1000 dilution. After washing, the membrane was incubated for 1.5 hours with the respective secondary antibodies at a 1:2000 dilution. Protein expression was visualized with the ECL technique (Amersham ECL Select). Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): YAP (4912S), TEAD1 (12292S), and c-Myc (9402S).
5
Cell Cycle Regulation and Apoptosis Analysis
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Reagents and antibodies. Dimethyl sulfoxide (DMSO), phosphate-buffered saline (PBS), trypsin-EDTA, fetal bovine serum (FBS), Roswell Park Memorial Institute (RPMI)-1640 medium, and penicillin-streptomycin were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). A cell cycle assay kit was purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Mouse or rabbit monoclonal antibodies specific for cyclin dependent kinase 4 (CDK4; 2906S), cyclin D1 (2978), c-myc (9402S), B-cell lymphoma 2 (Bcl-2; 2876S), Bcl-2 associated X protein (BAX; 2772S) and α-actin (4967L) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Bicinchoninic acid (BCA) protein assay and Enhanced Chemiluminescence (ECL) kits were obtained from Pierce; Thermo Fisher Scientific, Inc. All other chemicals used were of the highest grade commercially available.
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