B16, HepG2, A549, SW620, MCF-7 and Hela cells were obtained from ATCC and maintained by State Key Laboratory of Biotherapy, Sichuan University in DMEM medium supplemented with 10% fetal bovine serum (FBS, GIBCO, Australia). MTT was purchased from Sigma-Aldrich. Antibodies against hsp90α, stat3 were purchased from Genetex. Antibodies against Bcl-2, Bim, caspase-3, caspase-8, caspase-9, FasL, p27, c-myc, N-cadherin, EGFR, pEGFR(T1068), pEGFR(T1173), C-Raf, pC-Raf, ERK1/2, pERK1/2, Akt, pAkt(S308), pAkt(S473), P90RSK, pP90RSK, Met, pMet, PI3K, B-Raf, pB-Raf, hif1-α were purchased from Cell Signaling Technology. Antibodies against poly polymerase (PARP), FasL, JNK, pJNK, Met, pMet were purchased from Abcam. Antibodies against p21, β-actin, cytochrome c, Bad, Bax, E-cadherin, vimentin, MMP2, MMP9, ZEB1, β-catenin, hsp90β, hsp70, p53, mdm2, cdk2, cdk4, cdk6, cdc37, MNK1, ERK5, GAPDH and Secondary antibodies (HRP-conjugated sheep anti-rabbit antibodies or HRP-conjugated sheep anti-mouse antibodies) for western blot were obtained from Proteintech. Protein A/G Mix Magnetic Beads for Immunoprecipitation were purchased from Millipore. The detailed synthesis, characterization and in vitro biological procedures of compounds 8a–n were described in Supplementary materials .
B raf
Manufactured by Cell Signaling Technology
Sourced in United States
B-Raf is a lab equipment product that is a serine/threonine-protein kinase involved in regulating the MAP kinase/ERK signaling pathway. It plays a role in cell division, differentiation, and secretion.
Automatically generated - may contain errors
Lab products found in correlation
C raf, by Cell Signaling Technology (8 mentions)
P erk1 2, by Cell Signaling Technology (6 mentions)
Erk1 2, by Cell Signaling Technology (6 mentions)
β actin, by Cell Signaling Technology (6 mentions)
Phospho erk, by Cell Signaling Technology (4 mentions)
Mek1 2, by Cell Signaling Technology (4 mentions)
P braf, by Cell Signaling Technology (3 mentions)
P erk, by Cell Signaling Technology (3 mentions)
C myc, by Cell Signaling Technology (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Raf 1, by Cell Signaling Technology (2 mentions)
Total erk, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
P b raf ser445, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Phospho mek1 2, by Cell Signaling Technology (2 mentions)
Actin, by Cell Signaling Technology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Hsp70, by Proteintech (1 mentions)
21 protocols using b raf
1
Screening of Novel Compounds in Cancer Cell Lines
2
Protein Analysis of MAPK Signaling Pathway
3
Western Blot Analysis of Protein Targets
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Total proteins were extracted from cultured cells using radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific). The proteins were separated by 10% SDS‐PAGE and electroblotted onto polyvinylidene fluoride membranes (Merck KGaA) at 300 mA for 60 minutes. After blocking with 3% skim milk at room temperature for 1 hour, the membranes were incubated with primary antibodies at the appropriate concentrations at 4°C overnight: ENO1 (1:1000; #3810, Cell Signaling), ENO2 (1:1000; #9536, Cell Signaling), BRAF (1:1000; #14814, Cell Signaling), FOSL1 (1:1000; #D80B4, Cell Signaling), pERK (1:2000; #4370, Cell Signaling), ERK (1:1000; #4695, Cell Signaling), pAKT (1:2000; #4060, Cell Signaling), and AKT (1:1000; #9272, Cell Signaling). After incubation with secondary antibodies, protein bands were detected using the Amersham Enhanced Chemiluminescence Prime Western Blotting Detection Reagent (GE Healthcare).
4
Autophagy Modulation with Pharmacological Inhibitors
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Thioridazine (10-[2-(1-methyl-2-piperidyl) ethyl]-2-methyl-thiophenothiazine) (Figure 1 A), 3-methyladenine, leupeptin, bafilomycin A1 and chloroquine were purchased from Sigma-Aldrich, St. Louis, MO, USA. Rapamycin was obtained from Selleck Chemicals, Houston, TX, USA. LY294002 was obtained from Cell Signaling Technology, Danvers, MA, USA. Hoechst 33,258 and LysoTracker Green were obtained from Thermo Fisher Scientific, Waltham, MA, USA. Antibodies against AKT (pan) (#4691), Ambra-1 (#24907), AMPKα (#95832), Atg5 (#12994), Atg7 (#8558), BAK (#3814), BAX (#5023), BCL-xL (#2764), BCL-2 (#15071), BIM (#2933), B-Raf (#14814), caspase-3 (#9665), cleaved caspase-3 (Asp175) (#9661), cleaved caspase-8 (#9496) (Asp391), Lamp2 (#49067), LC3A/B (#12741), mTOR (#2983), NOXA (#14766), p-AKT (Ser473) (#4060), p-AMPKα (Thr172) (#2535), p-Beclin-1 (Ser15) (#13825), p-B-Raf (Ser445) (#2696), p-ERK1/2 (Thr202/Tyr204) (#9101), p-MEK1/2 (Ser217/221) (#9154), p-mTOR (Ser2448) (#2971), p-PI3K (Tyr199/458) (#4228), Ras (#3965), SQSTM1/p62 (#8025), ULK (#8054), β-actin (#3700), anti-mouse IgG HRP-linked (#7076), and anti-rabbit IgG HRP-linked (#7074) were obtained from Cell Signaling Technology, Danvers, MA, USA. Antibodies against Beclin-1 (612112), BID (611528), MCL-1 (559027) and PI3K (610045) were from BD Biosciences, San Jose, CA, USA.
5
Protein Expression Analysis in MM Cells
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MM cells were cultured with or without novel or conventional agents; cells were then harvested, washed, and lysed as reported [51 (link), 52 (link)]. Cell lysates were subjected to SDS-PAGE, transferred to membranes, and immunoblotted with the following antibodies: B-Raf, C-Raf, phospho-MEK1/2, MEK1/2, phospho-ERK, ERK, phospho-v-akt murine thymoma viral oncogene homolog (AKT), AKT, cleaved caspase-3, poly (ADP-ribose) polymerase (PARP), phospho-forkhead box O3 (FOXO3), FOXO3, B-cell lymphoma 2 (Bcl-2) interacting mediator of cell death (Bim), cellular myelocytomatosis (c-Myc), CCAAT-enhancer-binding protein homologous protein (CHOP), and β-Actin (all from Cell Signaling, Beverly, MA, USA). Protein expression was quantified using ImageJ (National Institutes of Health, Bethesda, MD, USA).
6
Apoptotic Pathways Activation Assay
7
Western Blot Signaling Pathway
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Bim, Bax, Cleaved PARP, BRAF, pCRAF (S338), CRAF, MEK, pMEK (S217/221), EGFR, pEGFR (Y1068), AKT, pAKT (S473), ERK, pERK (T202/Y204), AURKA, pAURKA, S6, pS6 (S240/244), and HRP–conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Actin was obtained from Merck Millipore (Darmstadt, Germany). The immunoblots were detected by SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Massachusetts, USA).
8
Western Blot Protein Analysis Protocol
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Whole cell lysates were collected from samples in urea-SDS buffer (8 M urea,1% SDS, 60 mM Tris (pH 6.8), 10% glycerol, and 5% ß-mercaptoethanol) after PBS wash, and sheared with a syringe and a 25g needle. Protein concentrations were determined by the BCA assay (Thermo Fisher Scientific). Lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membrane was blocked with 5% non-fat dry milk for 1 hour at room temperature and followed by incubation with the primary antibody solution overnight at 4 °C or 1 hour at room temperature. After a series of PBS washes, the membrane was incubated with HRP-linked secondary antibodies for 1 hour at room temperature. Protein bands were imaged using LI-COR Odyssey FC (LI-COR Biosciences). Densitometric analysis was performed on blots using LI-COR Image studio software. The following primary antibodies were used: BRAF (#9433), p44/42 MAPK (#9107), P-p44/42 MAPK (#4370), and p16 (#80772) from Cell Signaling Technology, BRAFV600E (ab200535) from Abcam, Dsg1 (#32–6000) from Thermo Fisher Scientific, GAPDH (G9545) from MilliporeSigma, and Tubulin (12G10) from Developmental Studies Hybridoma Bank. Secondary antibodies for immunoblotting were goat anti-mouse and goat anti-rabbit peroxidase (SeraCare Life Sciences).
9
Immunoprecipitation of Raf-Isoform Complexes
10
Western Blot Analysis of EGFR Signaling
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Cells were lysed in RIPA buffer (Tris pH 7.4 50 mM, NaCl 150 mM, NP-40 1%, SDS 0.1%, EDTA 2 μM) containing proteinase inhibitors (Roche) and phosphatase inhibitors (Roche). The cell lysates (20 μg protein) were subjected to SDS-PAGE and Western blot. Antibodies against the following proteins were used: phospho-EGFR (Y1068), phospho-EGFR (Y845), EGFR, phospho-MEK1/2 (S217/221), MEK1/2, phospho-ERK (T202/Y204), ERK, phospho-AKT (S473), AKT, ERBB3, INSR, DUSP4, DUSP6, Rab11, E-Cadherin, PTEN, AXL, HER2, CRKL, MET, IGF-1R, ARAF, BRAF, phospho-CRAF (S338), CRAF, NRAS, and Actin (Cell Signaling Technology).
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