The denatured protein samples (10 μg) were subjected to SDS/PAGE gel electrophoresis on 4–12% polyacrylamide gels (BioRad) and transferred electrophoretically onto nitrocellulose membranes. The membranes were then blocked with Odyssey blocking buffer (LI-COR), and probed overnight at room temperature with primary antibodies; phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) or total p44/42 MAPK (ERK1/2) (Cell Signaling Technology), used at 1:1,500–2,000 dilution. Separate immunoblots were done for total and phospho-ERK. After washing, blots were incubated with infrared fluorescence-conjugated secondary antibodies used at 1:5,000 for the detection of all primary antibodies. Both blocking buffer and IR dye-coupled secondary antibodies were obtained from LI-COR (Lincoln, NE). Fluorescence was imaged and quantified using a LI-COR Odyssey Imaging System.
Phospho p44 42 mapk erk1 2 thr202 tyr204
Manufactured by Cell Signaling Technology
Sourced in United States, China, United Kingdom
Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) is a lab equipment product that detects phosphorylation of p44/42 MAPK (Erk1/2) at Thr202 and Tyr204.
Automatically generated - may contain errors
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97 protocols using phospho p44 42 mapk erk1 2 thr202 tyr204
1
Western Blot Analysis of ERK Phosphorylation
2
Immunoblot Analysis of Signaling Pathways
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At the end of the experiments, HIT-T15 cells were lysed in RIPA buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1% NP40, 0.1% SDS, supplemented with protease and phosphatase inhibitors), and protein concentrations were determined using the BCA Protein Assay Kit. Thirty micrograms of total cell lysate was separated on a SDS-PAGE and transferred onto nitrocellulose. Filters were blocked in 5% BSA and incubated overnight at 4°C with primary specific antibodies (anti-GIP receptor (H-70) and anti-GAPDH (FL-335) from Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA; anti-phosphoAKT (Ser473), -PI3 Kinase (19H8), and -phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) from Cell Signaling Technology, Beverly, MA, USA). Secondary specific horseradish-peroxidase linked antibodies were added for 1 hour at room temperature. Bound antibodies were detected using an enhanced chemiluminescence lighting system (Luminata Classico, Millipore, Billerica, MA, USA), according to manufacturer's instruction. To verify equal loading of the proteins, membranes were stripped, reblocked, and reprobed to detect GAPDH. Values of proteins of interest were normalized to total amounts of GAPDH. Bands of interest were quantified by densitometry using the Alliance software. Results were expressed as percentages of CTR (defined as 100%).
3
Evaluating MAPK Activation in Liver Cancer
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PLC/PRF/5 and HepG2 cell lines were cultured in presence of regorafenib, sorafenib, GSK1838705A and ramucirumab alone or in combination for 48 h. According to the western blotting protocol above described, the levels of the protein investigated were evaluated with the following antibodies: of Phospho-p44/42 MAPK(Erk1/2) (Thr202/Tyr204) and p44/42 MAPK(Erk1/2), β-Actin (13E5) (Cell Signaling). Muse MAPK Activation Dual Detection Kit (Millipore), was used to measure total levels of ERK. This kit included a phospho-specific antiphospho-Erk1/2 (Thr202/Tyr204, Thr185/Tyr187)-Phycoerythrin and an anti-Erk1/2-PECy5 conjugated antibody, to measure the amount of MAPK phosphorylation relative to the total MAPK expression in the cells processed according to the user’s guide. The levels of both the total and phosphorylated protein can be evaluated in the cell in the same time, resulting in a normalized measurement of MAPK activation after treatments. PLC/PRF/5 and HepG2 cell lines were cultured in presence of Regorafenib, Sorafenib, GSK1838705A and Ramucirumab alone or in combination for 15 minas it was done in the experiments previously described. Results were representative of three independent experiments.
4
Western Blot Analysis of Cell Signaling
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Proteins from total cell lysates or tumor tissues were separated by standard SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were washed, blocked and incubated with primary antibodies against p44/42 MAPK (Erk1/2, #4695), phospho-p44/42 MAPK (Erk1/2, Thr202/Tyr204, #4370), p38 MAPK (#9212), phospho-p38 MAPK (Thr180/Tyr182, #9211), SAPK/JNK (#9258), phospho-SAPK/JNK (Thr183/Tyr185, #9211), caspase-3 (#9665), cleaved PARP (Asp214, #9544) and PARP (#9542) all purchased from Cell Signaling Technology (Boston, MA, USA). The primary antibody against Dragon (AF3597, AF3630), and the anti-sheep (HAF016) or anti-goat (HAF109) secondary antibodies were purchased from R&D Systems (Minneapolis, MN, USA).
5
Western Blot Analysis of Cellular Proteins
6
Quantitative Analysis of Signaling Proteins
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Western Blots were performed with 20 μg protein from each sample. After 48 hours transfection, WAC3CD5+ cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.2% SDS, 1% Triton X-100, 2 mM EDTA). Protein lysates were separated on 10% SDS-PAGE and blotted onto a 0.2 μm nitrocellulose membrane (Bio-Rad, Hercules, CA). The primary antibodies used were for anti-MADD (1:2000; Abcam), -PIK3R2 (1:1000; Abcam), -phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (1:1000; Cell Signaling), -p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling), -phospho-Akt (Thr308) (1:2000; Cell Signaling), -phospho-AKT (Ser473) (1:2000; Cell Signaling), -AKT (1:1000; Cell Signaling) and anti-actin (1:5000; Sigma-Aldrich, USA) at 4°C overnight. Then membranes were washed and incubated with anti-rabbit or anti-mouse horseradish peroxidase conjugate secondary antibody at room temperature for 1 hour. ECL plus Western blotting detection reagent was used for the detection of protein signals with X-ray film (Amersham Biosciences, Buckinghamshire, UK). Protein bands were quantified using densitometry as measured by Quantity One 4.6.2 software (Bio-Rad); hence relative protein expression was expressed in comparison to corresponding control.
7
Benzo[a]pyrene-induced MAPK Activation
8
Immunofluorescence Staining of Phosphorylated Proteins
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Immediately following the completion of a stimulation protocol, 16% paraformaldehyde (PFA) was added to each well to a final concentration of 4%, and cells were incubated in PFA in the dark for 10 min. Cells were then permeabilized with 100/50 μL (for 96/384-well plates) with phosphate buffered saline (PBS) + 0.1% Triton-X for 10 min. Cells were then further permeabilized with ice cold methanol for 10 min. After permeabilization, cells were blocked with 1% BSA at room temperature for 30 min. Primary antibody was diluted in PBS + 1% BSA according to the manufacturer's recommendation for immunofluorescence (phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), Cell Signaling #4370, 1:400 dilution; phospho-Akt (Ser473), Cell Signaling Technologies #9271, 1:800 dilution). 96/384-well wells were incubated with 50/25 μL of antibody dilution for 2 hr at room temperature (RT). Samples were incubated at room temperature in primary antibody (for two hours, after which primary antibody was removed and samples underwent five washes in PBS + 0.1% TWEEN-20 (PBS-T). Cells were then incubated with secondary antibody (Jackson Immunoresearch Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H+L)) and DAPI (ThermoFisher, #D1306, 300 nM) in PBS-T + 0.1% BSA for 1 hour at RT. Secondary antibody was removed, samples underwent 5 washes with PBS-T. Samples were imaged in PBS-T.
9
Activation of StMPK7 by CA-StMKK1
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GFP‐StMPK7 was coexpressed with CA‐StMKK1‐myc or the control GUS‐myc by transient transformation of N. benthamiana leaves. Proteins were extracted using an extraction buffer with phosphatase inhibitor cocktails 2 and 3 (Sigma). The activation of StMPK7 through phosphorylation was detected using anti‐pERK antibody (Phospho‐p44/42 MAPK [Erk1/2] [Thr202/Tyr204]; Cell Signaling Technology).
10
Immunoblot Analysis of Signaling Pathways
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