Proteins extraction and blotting was performed as in (24 (link)). The primary antibodies for phospho-ERK, total-ERK, phospho-Histone 3, Mcl-1, BIM and total MEK, were from Cell Signaling Technology. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Sigma Aldrich) was used as a loading control to demonstrate even protein loading. The secondary antibodies; goat anti-rabbit IgG HRP and sheep anti-mouse IgG HRP were from Amersham/ GE Healthcare. For mouse xenograft experiments, tumor samples were harvested and immediately stored in RNAlater solution (Ambion) at 4°C before protein extraction.
Total mek
Manufactured by Cell Signaling Technology
Sourced in United States
Total MEK is a lab equipment product that measures the total amount of mitogen-activated protein kinase kinase (MEK) in a sample. MEK is a key enzyme involved in the regulation of cellular signaling pathways. The Total MEK product provides a quantitative assessment of the overall MEK levels in a given biological sample.
Automatically generated - may contain errors
Lab products found in correlation
Total erk, by Cell Signaling Technology (7 mentions)
Total akt, by Cell Signaling Technology (6 mentions)
Total erk1 2, by Cell Signaling Technology (3 mentions)
P mek, by Cell Signaling Technology (3 mentions)
Phospho akt, by Cell Signaling Technology (3 mentions)
Phospho erk, by Cell Signaling Technology (2 mentions)
β catenin, by Merck Group (2 mentions)
Phospho mek s217 221, by Cell Signaling Technology (2 mentions)
Phospho erk t202 y204, by Merck Group (2 mentions)
β actin, by Merck Group (2 mentions)
P mek ser217 221, by Cell Signaling Technology (2 mentions)
Cox 2, by Cell Signaling Technology (2 mentions)
Phospho akt ser473, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Phospho mek, by Cell Signaling Technology (2 mentions)
Sheep anti mouse igg hrp, by Cytiva (1 mentions)
Rnalater solution, by Thermo Fisher Scientific (1 mentions)
Gapdh antibody, by Merck Group (1 mentions)
Goat anti rabbit igg hrp, by Cytiva (1 mentions)
Mcl 1, by Cell Signaling Technology (1 mentions)
15 protocols using total mek
1
Protein Extraction and Immunoblotting
2
Western Blot Analysis of Protein Expression
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Cells were lysed in Laemmli buffer and analyzed for total protein concentration as described (Karni et al. 2007 (link)). Fifty micrograms of total protein from each cell lysate was separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked with 5% milk and probed with specific antibodies. Bands were visualized using enhanced chemiluminescence detection. Primary antibodies are as follows: hnRNP A1 (mAb A1/55, 1:1000) (Allemand et al. 2005 (link)), hnRNP A2/B1 (1:1000, Santa Cruz), β-tubulin (1:1000, Sigma), β-catenin (1:2000, Sigma), β-actin (1:1000, Santa Cruz), T7 tag (1:5000, Novagen), phospho-MEK S217/221 (1:1000, Cell Signaling), total MEK (1:1000, Cell Signaling), phospho-ERK T202/Y204 (1:1000 Sigma), total ERK1/2 (1:1000, Cell Signaling), A-Raf (1:1000, Cell Signaling), and A-Raf short (1:500) (Rauch et al. 2011 (link)). Secondary antibodies are as follows: HRP-conjugated goat anti-mouse, goat anti-rabbit, donkey anti-goat IgG (H+L; 1:10,000 Jackson Laboratories).
3
Immunoblotting of Cellular Signaling Proteins
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The cells were washed two times with ice-cold PBS and collected with the cell lysis buffer (10 mM Tris-HCl, pH 7.4, 1% SDS and 1 mM Na3VO4). The cell extracts were sonicated, denatured by heating at 100 °C for 5 min and quantified with a Dc protein assay kit (Bio-Rad, Hercules, CA, USA). Equal aliquots of cell extracts were separated on SDS-PAGE. The proteins were then transferred to PVDF membranes (Bio-Rad), blocked and probed with one of the antibodies against phospho-S6 S235/236, phospho-ERK T202/Y204, phospho-MEK1/2 S217/221, phospho-c-Raf S388, S289/296/301, phospho-p90RSK S359/363, S380, S573, total-ERK, total-MEK, total-c-Raf, total-p90RSK, total-S6, Ras and GAPDH (Cell Signaling Technology), p27 for C-terminal (Abcam), HIF-1α and PHLPP (Bethyl Laboratories Inc., Montgomery, TX, USA), p27 for N-terminal and HA (Santa Cruz Biotechnology) or β-actin (Sigma). Primary antibody-bound proteins were detected by using an alkaline phosphatase-linked secondary antibody and an ECF Western Blotting System (Amersham, Piscataway, NJ, USA).
4
Western Blot Analysis of Signaling Proteins
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Protein extracts were run on an 8% SDS-PAGE gel, transferred to Immobilon-P transfer membranes (Millipore Corp., Bedford, MA), and visualized with antibodies (Abs) specific for GAB1 (Cell Signaling), p-MEK (Ser217/221; Cell Signaling), total MEK (Cell Signaling), p-AKT (Thr 308; Cell Signaling), total AKT (Cell Signaling), IE86 (monoclonal antibody [MAb] 810; Millipore), UL138 (14 (link)), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Abcam). Relative levels of intensity of bands detected by Western blotting were quantitated using ImageJ software. In each case, the relative intensity of each band was compared to that of the control protein (total MEK or total AKT [Fig. 2 ] or GAPDH [Fig. 1 , 3 , and 4 ]). The ratio of sample to control was set to a value of 1 for the mock treatment (Mock) or the first time point, and each subsequent sample/control ratio is presented as a multiplier of the reference time point.
5
Quantification of MAPK Signaling
6
Biochemical Signaling Pathway Analysis
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1,8-cineole (99%) and N-Acetyl-L-cysteine (NAC, 99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Antibodies specific to detect Ser217/221-phospho MEK, total MEK, Ser257/Thr261-phospho MKK4/7, total MKK4/7, Ser178/207-phospho MKK3/6, total MKK3/6, Thr202/Tyr204-phospho ERK, total ERK, Tyr180/182-phospho p38, total p38, Thr183/Tyr185-phospho JNK, total JNK, Ser455-phospho BRAF, Ser338-phospho CRAF, Tyr416-phospho Src, Tyr845 and Tyr1068-phospho EGFRs, total EGFR, Ser473-phospho Akt, total Akt, COX-2, Ser133-phospho CREB, and CREB were obtained from Cell Signaling Biotechnology (Beverly, MA, USA). Antibodies specific to total BRAF (F-7), total CRAF (C-12), AhR (H-211), c-Src (B-12), and β-actin (C-4) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The protein assay kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA).
7
Quantification of Signaling Proteins
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Antibodies against total ERK, phospho-ERKthr202/tyr204, total MEK, phospho-MEKser217/221, phospho-p70-S6 kinasethr389, phospho-RPS6ser235/236, cleaved PARP, B-actin, phospho-4EBP1thr37/46, total AKT, phospho-AKTser473, and phospho-AKTthr308 were obtained from Cell Signaling Technology, NF1 (A300-140A) from Bethyl, and Cyclin D1 from Santa Cruz Biotechnology. Trametinib and sapanisertib were purchased from SelleckChem. Drugs for in vitro studies were dissolved in DMSO to yield 10 mM or 1 mM stock solutions and stored at −20°C.
8
Western Blot Analysis of Cell Signaling
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Cells were lysed in Laemmli buffer and analyzed for total protein concentration. 20 μg of total protein from each cell lysate was separated by SDS-PAGE and transferred on to a PVDF membrane. The membranes were blocked with 5% milk and probed with specific antibodies. Bands were visualized using enhanced chemiluminescence detection. Primary antibodies: β-Catenin (1:2000, Sigma), Phospho-MEK S217/221 (1:1000, Cell Signaling), total MEK (1:1000, Cell Signaling), phospho-ERK T202/Y204 (1:1000 Sigma), total ERK1/2 (1:1000, Cell Signaling), phospho-AKT (1:1000, Cell Signaling), AKT (1:1000, Cell Signaling), cleaved Caspase 3 (1:1000, Cell Signaling) SRSF1 (AK96 culture supernatant 1:300), CUGBP1 (1:1000, Santa Cruz Biotechnology), MBNL1 (1:250, Santa Cruz Biotechnology), SRSF3 (1:1000, Santa Cruz Biotechnology). Secondary antibodies: HRP-conjugated goat anti-mouse, goat anti-rabbit and donkey anti-goat IgG (H+L; 1:10000 Jackson Laboratories).
9
MAPK Signaling Pathway Activation
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HCT-116 and LOVO cells (ATCC) were maintained in DMEM or DMEM/F12 respectively supplemented with antibiotics, 10% FBS, and 2 mM glutamine. Cells were seeded in 100 mm plates for assays. After 24 hours, cells were treated with 1 or 10 for 1 hour. Cells were then harvested in PBS and lysed in buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% Sodium Cholate, 0.1% SDS) containing protease and phosphatase inhibitors (Thermo Fisher Scientific). Cell lysates were spun at 20,000 rpm for 20 min and supernatants were extracted, normalized for total protein based on the BCA assay (Thermo Fisher Scientific), and mixed with 6× SDS dye prior to application to a 4–15% Tris-HCl gradient gels. Following PAGE separation, samples were transferred to nitrocellulose and blotted for the indicated proteins using antibodies for pMEK (#9154), pERK1/2 (#9101), and total MEK (#8727) (all from Cell Signaling Technology; Danvers, MA).
10
Immunoblotting of Melanoma Cell Signaling
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All melanoma cells were washed with ice-cold phosphate buffered saline (PBS) twice and lysed with RIPA buffer containing phosphatase and protease inhibitors (all from Sigma Aldrich, St. Louis, MO). Protein extracts were separated with SDS-PAGE in 4-12% tris-glycine gels and transferred to immun-blot PVDF membrane. After blocking for 1 hour in PBS containing 0.1% Tween 20 and 5% nonfat milk or 5% bovine serum albumin (BSA) in PBS, the membrane was exposed to various primary antibodies overnight, followed by secondary antibodies conjugated to horseradish peroxidase. ECL-Plus kit (Amersham Biosciences Co, Piscataway, NJ) was used to check immunoreactivity and blots were scanned using a Typhoon scanner (Amersham Biosciences Co.). Primary antibodies included pERK Thr202/Tyr204, total ERK, pMEK Ser217/221, total MEK, pAKT Ser473, total AKT, pRSK, total RSK, beta-actin (all from Cell Signaling Technology, Danvers, MA).
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