Western blot analysis was conducted as described previously45 (link). Briefly, MLE-12 cells were lysed using RIPA buffer containing 1 mM of PMSF, 1 μg/mL of aprotinin, 1 μg/mL of leupeptin, 1 mM of Na3VO4, and 1 mM of NaF, and then stored in aliquots at − 80 °C until further analysis. The lysate (20 μg) was mixed with an equal volume of sample buffer, denatured by boiling, and then separated on 10–15% polyacrylamide mini-gel. The proteins were transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL), blocked with 5% milk, and incubated overnight with E-cadherin (Abcam, Cambridge, MA, ab11512), C-Raf (Abcam, ab137435), phospho-C-Raf (Abcam, ab60985), Twist1 (Abcam, ab49254), and ɑ-SMA (Abcam, ab21027).
C raf
Manufactured by Abcam
Sourced in United States
C-Raf is a serine/threonine-protein kinase that is involved in the MAPK/ERK signaling pathway. It acts as a mediator of the Ras signal transduction pathway.
Automatically generated - may contain errors
Lab products found in correlation
Erk1 2, by Abcam (2 mentions)
Mek1 2, by Abcam (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
E cadherin, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Vascular endothelial growth factor (vegf), by Abcam (1 mentions)
Hif 1α, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Fdbio (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Novablot system, by Cytiva (1 mentions)
Gapdh, by Proteintech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Abcam (1 mentions)
Caspase 3, by Abcam (1 mentions)
Caspase 9, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Cyclin b1, by Abcam (1 mentions)
Bcl xl, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
P erk1 2, by Abcam (1 mentions)
3 protocols using c raf
1
Western Blot Analysis of EMT Markers
2
Molecular Signaling Pathways Modulation
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After treatment with siRNA negative control (si-NC), si-Spry3, agomiR-NC, agomiR-18a, agomiR-BART10-5p, agomiR-18a combined agomiR-BART10-5p, antagomiR-NC, antagomiR-18a, antagomiR-BART10-5p, and antagomiR-18a combined antagomiR-BART10-5p, HONE1 and HONE1-EBV cells were harvested and lysed in lysis buffer supplemented with protease inhibitors. MicroRNA mimics, agomiRs, inhibitors, and antagomiRs were transfected at 50 nmol/L with Lipofectamine 2000 reagent (Invitrogen, USA). Protein lysate was resolved on 10% SDS-PAGE followed by blot transfer onto a polyvinylidene fluoride (PVDF) membrane (Millipore, USA) using the semidry NovaBlot system (Amersham Pharmacia, UK) for western blot analysis. After that, the membranes were first incubated with antibodies against GAPDH (Proteintech, USA), Spry3, Ras, c-Raf, MEK1/2, Erk1/2, mTOR, eIF4E1, VEGF, mmp2, and HIF1-α (Abcam, UK) overnight at 4°C, followed by a 1- to 2-h incubation with horseradish peroxidase-conjugated secondary antibody. The protein signals were detected using an enhanced chemiluminescence kit (Fdbio Science, China) and analyzed using the Bio-Rad (USA) imaging system and the associated software according to the manufacturer’s instructions. Antibodies concentrations are listed in Table S1 .
3
Spiclomazine Modulates Cell Signaling Pathways
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Media (DMEM and IMDM) and fetal bovine serum (FBS) were purchased from Gibco (Grand island, NY). Unless otherwise notified, all chemicals were obtained from Sigma-Aldrich. Spiclomazine, 1-Thia-4,8-diazaspiro[4.5]decan-3-one,8-[3-(2- chloro-10H-phenothiazin-10-yl)propyl]-, hydrochloride (1:1) (Supplementary Figure 1 ), was kindly supplied from NCI/DTP Open Chemical Repository (http://dtp.cancer.gov ) and further confirmed. Spiclomazine was dissolved in DMSO to make stock solution (10 mg/mL) and further diluted to appropriate concentrations with double distilled water containing 10% DMSO immediately before use. The c-Raf, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, Caspase-3, Caspase-9, Bcl-xl, Bcl-2, Bax, β-Actin, CDK1 and Cyclin B1 primary antibodies were purchased from Abcam (Cambridge, MA). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse secondary antibodies were obtained from BD Bioscience (San Jose, CA).
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