AHSA1, N-cadherin, 6-his, vimentin, HSP90, MEK1/2, E-cadherin, and CALD1 antibodies were obtained from Proteintech (Wuhan, China). ERK1/2, Phospho-MEK1/2, and Phospho-ERK1/2 antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Ki67 antibody was obtained from Abcam (Cambridge, MA, USA). Phospho-CALD1 antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA). β-tubulin antibody was obtained from Bioworld Technology (Bloomington, MN, USA).
Mek1 2
Manufactured by Proteintech
Sourced in United States
MEK1/2 is a lab equipment product manufactured by Proteintech. It is a dual-specificity protein kinase that plays a central role in the ERK/MAPK signaling pathway. MEK1/2 acts as a critical upstream regulator of the ERK1/2 kinases, which are involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Erk1 2, by Proteintech (2 mentions)
Phospho mek1 2, by Cell Signaling Technology (2 mentions)
Ki67 antibody, by Abcam (1 mentions)
E cadherin, by Proteintech (1 mentions)
N cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
Hsp90, by Proteintech (1 mentions)
6 his, by Proteintech (1 mentions)
Cald1, by Proteintech (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Boster Bio (1 mentions)
Ripa buffer, by Boster Bio (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Impdh2, by Proteintech (1 mentions)
Fanci, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Proteintech (1 mentions)
Enhanced chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Vascular endothelial growth factor (vegf), by Proteintech (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
4 protocols using mek1 2
1
Antibody Panel for Cell Signaling Analysis
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells using RIPA buffer (Boster, Wuhan, China) containing the protease inhibitor PMSF (Boster). Proteins were resolved by SDS-PAGE and transferred to PVDF membranes. The blots were blocked by incubation with 5% fat-free milk at room temperature for 2 h and then incubated overnight at 4°C with a 1:500 dilution of antibodies to the following proteins: FANCI (Santa Cruz Biotechnology, Dallas, TX, USA), IMPDH2, MEK1/2, ERK1/2, MMP2, MMP9, GAPDH (all Proteintech, Wuhan, China), phospho (p)-MEK1/2, and p-ERK1/2 (both Cell Signaling Technology, Danvers, MA, USA). The membranes were washed three times with TBST and then incubated for 2 h with horseradish peroxidase-conjugated rabbit or mouse secondary antibodies. After signal development, expression of proteins was analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Quantitative Analysis of Apoptosis and Signaling Pathways
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Western blotting was used to detect the expression of proteins involved in apoptosis, angiogenesis and the MEK/ERK signalling pathway at 24 h after administration. Total protein was extracted using protein extraction kit, according to the manufacturer’s instructions and the protein concentration were measured with BCA method. Protein samples (40 μg) were subjected to 10% SDS-polyacrylamide gels electrophoresis (SDS-PAGE) to separate. Then transferred to poly-vinylidnene fluoride (PVDF, Millipore) membranes and blocked in PBST solution with 5% non-fat milk. The membranes were incubated in primary antibodies against brain derived neurotrophic factor (BDNF), nerve growth factor (NGF), Bax, Bcl-2, VEGF, FGF2, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2 (1:2000 dilution, Proteintech, USA) at 4 °C overnight. Secondary antibodies: HRP-conjugated anti-mouse or anti-rabbit IgG (1:5000, Proteintech, USA) were incubated 1 h at 37 °C. Finally, the enhanced chemiluminescent reagent (Thermo Fisher, USA) was added in the membranes and band intensity signals were observed. GAPDH was used as an internal reference, and the optical densities of protein bands were analysed by Image J software to represent the relative expression of target protein.
4
Western Blot Analysis of Protein Targets
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Cells were lysed in RIPA buffer (Pioneer, Shanghai, China) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (TargetMol, USA). Protein concentration determined using a BCA protein assay kit (Proteintech, Wuhan, China). The protein samples were transferred to PVDF membrane by SDS-PAEG, membranes were incubated with specific primary antibody at room temperature for 30 min and then overnight at 4 °C. Then they were incubated with corresponding anti-rabbit/anti-mouse secondary antibody (TransGen Biotech, Beijing) at room temperature for 1 h. The membranes were incubated with ECL (Boster Biological Technology co.ltd, California, USA) in the dark for chemiluminescence detection. Luminescent signals were detected and recorded by Syngene GBox (Syngene, Cambridge, UK). The primary and secondary antibodies used are listed as follows: RPL5 (Proteintech, 29,092–1-AP), MMP2 (Proteintech, 10,373–2-AP), MMP9 (Proteintech, 10,375–2-AP), CDK4 (Proteintech, 11,026–1-AP), CyclinD1 (Proteintech, 26,939–1-AP), MEK1/2 (Proteintech, 11,049–1-AP), p-ERK (Proteintech, 28,733–1-AP), ERK (Proteintech, 67,170–1-Ig), c-Myc (Proteintech, 10,828–1-AP), p-MEK1/2 (Cell Signaling Technology, 9154), FOXO3 (Boster, BM4734) and β-Tubulin (Proteintech, 66,240–1-Ig).
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