Cells were lysed in buffer (62.5 mmol/L Tris‐HCl, 2% SDS, 10% glycerol, 0.5 mmol/L PMSF and 2 µg/mL aprotinin, pepstatin and leupeptin), and the protein lysates were subjected to SDS‐PAGE followed by electroblotting onto a polyvinylidene difluoride (PVDF) membrane. Membranes were probed with monoclonal antibodies against FHL2 (1:500; Santa Cruz Biotechnology), p‐RAF1 (1:1000; Abcam), MEK1 (1:1000; Abcam), p‐MEK1 (1:1000; Abcam), ERK (1:1000; Abcam), p‐ERK (1:1000; Abcam), p38 (1:1000; Cell Signaling Technology), p‐p38 (1:1000; Cell Signaling Technology), Cyclin‐dependent kinase 2 (CDK2) (1:1000; Santa Cruz Biotechnology), Cyclin‐dependent kinase 4 (CDK4) (1:1000; Santa Cruz Biotechnology), cyclin E (1:1000; Santa Cruz Biotechnology), AKT (1:1000; Cell Signaling Technology), p‐AKT (1:1000; Cell Signaling Technology), PI3K p110β (1:1000; Abcam), Rac1 (1:1000; Abcam) and β‐actin (1:5000). Protein expression was visualized using chemiluminescence detection reagents (Merck Millipore). The results were analysed using ImageJ software.
P raf 1
Manufactured by Abcam
Sourced in United Kingdom
P-Raf-1 is a laboratory reagent used for research purposes. It is a phosphorylated form of the Raf-1 protein, which is a key component of the MAPK/ERK signaling pathway. P-Raf-1 can be used to study the regulation and function of this important cellular pathway.
Automatically generated - may contain errors
Lab products found in correlation
P erk1 2, by Abcam (3 mentions)
P mek1, by Abcam (2 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Chemiluminescence detection reagent, by Merck Group (1 mentions)
Cyclin dependent kinase 4, by Santa Cruz Biotechnology (1 mentions)
β actin, by Abcam (1 mentions)
Cyclin dependent kinase 2, by Santa Cruz Biotechnology (1 mentions)
Cyclin e, by Santa Cruz Biotechnology (1 mentions)
P erk, by Abcam (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
Total protein extraction kit, by Keygen Biotech (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Efficient chemiluminescence ecl kit, by Thermo Fisher Scientific (1 mentions)
Ab137435, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Ab 60985, by Abcam (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
6 protocols using p raf 1
1
Western Blot Analysis of Signaling Proteins
2
Western Blot Analysis of RAF1 and p-RAF1
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The total protein extraction kit (KeyGen BioTECH, Jiangsu, China) was used to isolate proteins from the above 10 patients. The protein lysates were separated by 10% sodium dodecyl sulphate—polyacrylamide gels (SDS-PAGE) (Beyotime, Shanghai, China), and then transferred onto polyvinylidene difluoride (PVDF) membranes (Beyotime, Shanghai, China). Primarry antibodies against RAF1, phospho-RAF1 (p-RAF1) and GAPDH were purchased from Abcam (ab-137435, ab-60985, ab-181602; Abcam, Cambridge, UK) and were diluted at 1:1000, 1:1000 and 1:3000. The secondary antibody goat anti-rabbit IgG (Beyotime, Shanghai, China) was diluted at 1:5000. The Efficient chemiluminescence (ECL) kit (Thermo, Shanghai, China) was utilized to detect horseradish peroxidase (HRP) and its products.
3
Western Blot Analysis of MAPK Signaling Pathway
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Western blot was performed as previously described (27 (link)). Samples of equivalent total protein (40 μg) were loaded. Primary antibody against REST, p-Raf-1, p-beta-catenin, p-MEK-2, p-ERK-1/2 (Abcam, Cambridge, UK, 1:500), E-cadherin, N-cadherin, Vimentin, MMP-9, ZO-1, MEK-2, ERK1/2, Raf-1, beta-catenin, beta-Actin, and Histone H3 (ProteinTech Group, Rosemont, USA, 1:500) were used. Suppression of MAPK signaling pathway with U0126 was performed as previously described (28 (link)). In brief, cells incubated in t25 culture flask with 60~70% confluency were treated with 10 μM U0126 for 36 h before further analysis.
4
Protein Extraction and Western Blot Analysis
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The total protein derived from each group of cells and from the rabbit carotid artery tissues was extracted by a protein extraction kit (Beijing solarbio science﹠technology, China). The process was conducted according to the kit instructions. The BCA (Bi Yuntian, China) protein assay kit was used to determine the protein concentration. A total of 40 μg protein samples were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked at room temperature with skimmed milk powder for 2 h and subsequently incubated with p-Raf-1, p-MEK, p-ERK1/2, Raf-1, MEK and ERK1/2 antibodies (Abcam, UK) at 4°C overnight. Subsequently, the membranes were incubated with the corresponding secondary antibody IgG (1:2000) for 1 h at room temperature.
5
Protein Expression Analysis by Western Blot
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The protein concentration was measured by using the BCA kit (ThermoFisher Scientific, USA). The protein sample was added with 5 × Loading buffer, boiled in water for 10 min, and then carried out sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) protein electrophoresis. After the electrophoresis, the protein was transfected to the polyvinylidene difluoride (PVDF) membrane, and blocked with 5% skim milk for 2 h at room temperature. The membranes were incubated with specific antibodies (Rabbit anti-Human, anti-GAPDH, Ras, p-Raf1, Raf1, p-MEK1, MEK1, p-ERK1/2, ERK1/2, 1:1000, Abcam) overnight at 4 °C. Then, membranes were incubated with the horseradish peroxidase-conjugated antibody (Goat anti-Rabbit IgG, 1:1000, Sigma) at room temperature for 1 h. ECL reagent (Beyotime) was used to detect the protein signals.
6
Lung SCC Cell Culture and Molecular Assays
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Human lung SCC cell SK-MES-1 was purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). NCI-H520 was a kind gift from Dr. Ying (Department of Respiratory Diseases, Sir Run Run Shaw Hospital, Zhejiang University, Hangzhou, China). Both cell lines were maintained in RPMI-1640 (Gibco BRL Co.Ltd., MD, USA), supplemented with 10% fetal bovine serum (FBS, Gibco BRL Co.Ltd., MD, USA), 100U/ml penicillin and 100μg/ml streptomycin (Gibco BRL Co.Ltd., MD, USA) at 37℃ with 5% CO2. Deguelin was obtained from Sigma (St. Louis, MO, USA). Galectin-1 shRNA plasmid, negative control shRNA plasmid, shRNA plasmid transfection reagent and shRNA plasmid transfection medium were purchased from Santa Cruz Biotechnology (CA, USA). Galectin-1 expression plasmid pCMV3-SP-N-His-Gal-1 and empty vector pCMV3-SP-N-His plasmid were from Sino Biological Inc. (Beijing, China). OPTI-MEM was purchased from Gibco BRL Co.Ltd. (MD, USA) and HiPerFect transfection reagent was obtained from Qiagen (Hilden, Germany). Antibodies against rabbit Galectin-1, H-Ras, Raf-1, p-Raf-1 and Raf kinase inhibitor L779450 were purchased from Abcam (Cambridge, MA, USA). GAPDH, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, phospho-p44/42 MAPK(T202/Y204), p44/42 MAPK antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).
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