Crude cell extracts were prepared by lysing cells using RIPA lysis buffer (Pierce) supplemented with phosphatase and protease inhibitor cocktails (Roche). The extracted proteins (20–50 μg) were resolved with 10% SDS polyacrylamide gels and transferred onto a nitrocellulose membrane (Millipore) according to standard protocols. Polyclonal anti-CXCR4 antibody (1:1000, Abcam), anti-c-kit antibody (1:500, Abcam), anti-CXCR4-phospho-serine 339 antibody (1:500, Abcam), anti-ERK1/2 antibody (1:1000, Cell Signalling Technology), anti-phospho-ERk1/2 antibody (1:1000, Cell Signalling Technology), anti-p38 antibody (1:1000, Cell Signalling Technology), anti-phospho-p38 antibody (1:1000, Cell Signalling Technology), anti-GRK6 antibody (1:1000, Santa Cruz), and anti-GRK2 antibody (1:1000, Santa Cruz) were used overnight at 4 °C. This was followed by incubation with a horseradish peroxidase-conjugated secondary antibody (1:10,000; Pierce). Peroxide activity was detected using the enhanced chemiluminescence Supersignal West Dura system (Pierce). As a loading control, mouse anti-beta-actin antibody was used at a concentration of 1:1,000.
Anti phospho erk1 2 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
The Anti-phospho-ERK1/2 antibody is a laboratory reagent used to detect the phosphorylated forms of Extracellular Signal-Regulated Kinase 1 and 2 (ERK1/2) proteins. ERK1/2 are key signaling molecules that play a crucial role in cellular processes such as proliferation, differentiation, and survival. This antibody specifically recognizes the phosphorylated residues of ERK1/2, allowing researchers to monitor the activation state of this important signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti erk1 2 antibody, by Cell Signaling Technology (13 mentions)
Anti gapdh antibody, by Cell Signaling Technology (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti phospho akt antibody, by Cell Signaling Technology (3 mentions)
Anti akt antibody, by Cell Signaling Technology (3 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti total erk1 2 antibody, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Cell Signaling Technology (2 mentions)
Anti tubulin antibody, by Cell Signaling Technology (2 mentions)
Anti n cadherin antibody, by Cell Signaling Technology (2 mentions)
Anti lamin b1 antibody, by Cell Signaling Technology (2 mentions)
Anti raf 1 antibody, by Cell Signaling Technology (2 mentions)
Anti hnrnpa2b1 antibody ab6102, by Abcam (2 mentions)
Anti snail antibody, by Cell Signaling Technology (2 mentions)
Hrp conjugated goat anti mouse, by Cell Signaling Technology (2 mentions)
Anti e cadherin antibody, by Cell Signaling Technology (2 mentions)
Anti hif 1α antibody, by Cell Signaling Technology (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
33 protocols using anti phospho erk1 2 antibody
1
Western Blot Analysis of CXCR4 and Signaling
2
Histological Analysis of Mouse Femurs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For histological analysis, 3- or 8-week-old femurs were fixed in 10% neutral formalin at 4 °C for 2 days and then decalcified in 15% tetrasodium EDTA (pH 8.0) at 4 °C for 2 weeks. Tissues were dehydrated in different concentrations of EtOH, incubated in xylene, and embedded in paraffin. Paraffin sections were performed at a 7-µm thickness along the coronal plate and stained with hematoxylin and eosin (H&E).
For immunohistochemistry, longitudinal sections of 8-week-old femurs were stained with anti-phospho-ERK1/2 antibody (Cell Signaling, 4376). Briefly, after deparaffinization and rehydration, paraffin tissue sections were blocked with 3% goat serum, 1% BSA, 0.1% Triton X-100 in PBS for 1 h at room temperature, incubated with primary antibody specific to phospho-ERK1/2 overnight at 4 °C, followed by TSA-biotin (Perkin Elmer, Waltham, MA, USA) and streptavidin-HRP, and then visualized with 2,2′-diaminobenzidine tetrahydrochloride as per the manufacturer’s instructions.
For immunohistochemistry, longitudinal sections of 8-week-old femurs were stained with anti-phospho-ERK1/2 antibody (Cell Signaling, 4376). Briefly, after deparaffinization and rehydration, paraffin tissue sections were blocked with 3% goat serum, 1% BSA, 0.1% Triton X-100 in PBS for 1 h at room temperature, incubated with primary antibody specific to phospho-ERK1/2 overnight at 4 °C, followed by TSA-biotin (Perkin Elmer, Waltham, MA, USA) and streptavidin-HRP, and then visualized with 2,2′-diaminobenzidine tetrahydrochloride as per the manufacturer’s instructions.
3
Antibody Characterization for Cardiac Research
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The primary antibodies used were anti-cofilin1 (Cell Signaling), custom-made anti-phospho(T25)-cofilin-1 (GenScript, dilution 1:50), anti-phospho(Ser3)-cofilin-1 (Cell Signaling), anti-phosphoERK1/2 antibody (Cell Signaling), anti-ERK1/2 (Cell Signaling), anti-MRTF-A (Santa Cruz Biotechnology), anti-Cx43 (Abcam), anti-α-tubulin (Abcam), anti-acetylated α-tubulin (Sigma), anti-Flag (Sigma), anti-GAPDH (Abcam), anti-N-cadherin (Cell Signaling), anti-Cx43 (Abcam), anti-actin (Cytoskeleton), anti-cardiac troponin T (Cell Signaling), anti-HDAC6 (Cell signaling), and anti-α-actinin sarcomeric (Sigma). The secondary antibodies for immunofluorescence were Alexa Fluor 488-conjugated goat anti-rabbit IgG, Alexa Fluor 568-conjugated goat anti-mouse IgG, and Alexa Fluor 488-conjugated donkey anti-goat IgG (Life Technologies). For immunoblotting, fluorescent-conjugated StarBright Blue 520 anti-mouse or StarBright Blue 700 anti-rabbit secondary antibodies (BioRad) were used. All the antibodies were used at dilutions recommended by the manufacturer.
4
Western Blot for Phospho-Erk1/2 Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fifteen microgram of protein lysate was loaded in each slot. SDS-PAGE was carried out at 20 mA for 1 h in a 12% bisacrylamid gel using a Mighty Small II Deluxe Mini Vertical Electrophoresis Unit (SE260, Hoefer, Holliston, USA). Protein was subsequently transferred to a PVDF membrane (88518, Thermo Scientific®, Waltham USA) using a Maxi-BlotTank fully wet blotter (340.000, GP Kunststofftechnik, Germany) at 60 mA for 4 h at 4°C. Membranes were subsequently stained with anti-phospho-Erk1/2 antibody (#9101 Cell Signaling Technologies) in 1:1000 dilution and goat anti rabbit horseradish peroxidase (HRP) conjugated (31460, Thermo Scientific®) in 1:10 K dilution as described in detail in Wiedemann et al. (2006 (link)). Signal detection was performed by enhanced chemi-luminescence using SuperSignal West Dura Chemilumenescent Substrate (37071, Thermo Scientific®) and CL-Xposure X-Ray films (34091, Thermo Scientific®). X-Ray films were subsequently scanned.
5
Recombinant Protein Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant mouse Src was expressed in Escherichia coli BL21 (DE3) and purified as described previously [3] (link). Recombinant human Abl1 and Lyn were purchased from Carna Biosciences. Anti-cAMP-dependent protein kinase (PKA) antibody, anti-phospho-PKA antibody, anti-extracellular-signal-regulated protein kinase (Erk)1/2 antibody and anti-phospho-Erk1/2 antibody were purchased from Cell Signaling Technology. Anti-Syk polyclonal antibody was obtained from Santa Cruz Biotechnology.
6
Oxidative Stress Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A fluorogenic dye, 2’,7’-dichlorofluorescin diacetate (H2DCF-DA), Hoechst 33342 and Rhod-2 AM were from Molecular Probes (Eugene, Oregon, USA). Fura-2/AM, Isopropanol and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were from Sigma-Aldrich (St. Louis, Missouri, USA). Anti- phospho-p38 and p38, PKG antibodies were purchased from Santa Cruz (Dallas, Texas USA); anti-eNOS, anti-iNOS, Bcl-2, Bax, and ERK 1/2 antibodies were obtained from Invitrogen (Waltham, Massachusetts, USA); anti- sGCα1 and β-actin antibodies from Sigma-Aldrich (St. Louis, Missouri, USA); anti- phospho-JNK and JNK antibodies were purchased from R&D System (McKinley Place NE, Minneapolis, USA); anti-phospho ERK 1/2 antibody was from Cell Signaling (Danvers, Massachusetts, USA).
7
Exendin-4 Signaling in Vascular Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Materials were purchased from Wako (Kyoto) or Nacalai Tesque (Kyoto) unless stated otherwise. Exendin-4 was procured from Sigma-Aldrich (St Louis, MO). The antibodies used for the western blot analyses were purchased as follows: anti SM22α, anti phospho-Yes-associated protein 1 (Yap1), anti-phospho-ERK1/2 antibody and anti-phospho-SAPK/JNK antibodies were purchased from Cell Signaling Technology, while ECL and ECL plus systems were purchased from GE Healthcare. Collagen I was purchased from Nippon Meat Packers, Inc. (Osaka). All chemical compounds were dissolved in dimethyl sulfoxide (DMSO) at final concentration of less than 1% except where specially noted.
8
Western Blotting of Cerebral Cortex Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting of brain tissues were performed as previously described [18 (link)]. The cerebral cortexes of mice were homogenized in lysis buffer (50 mM HEPES, pH7.2, 150 mM NaCl, 5 mM MgCl2) containing cOmplete™ EDTA-free protease inhibitor (Roche Diagnostics, Basel, Switzerland)) and centrifuged at 1,000 g for 5 min. The tissue lysates (20 μg of protein) were separated on SDS-PAGE (10% acrylamide gel). The separated proteins were transferred to a polyvinylidene difluoride membrane (Pall Life Sciences, Port Washington, NY). The membrane was blocked with 5% skim milk and incubated with an anti-ERK1/2 antibody (1:1000 dilution, BRID: AB_330744, Cell Signaling Technology, Danvers, MA), an anti-phospho-ERK1/2 antibody (1:1000 dilution, BRID: AB_331646, Cell Signaling Technology) or an anti-β-actin polyclonal antibody (1:1000 dilution, BRID: AB_476693, Sigma-Aldrich, St Louis, MO) overnight at 4 °C. The immunoreactive bands were visualized using a peroxidase-conjugated anti-rabbit IgG antibody (1:10000 dilution, BRID: AB_2337943, Jackson ImmunoResearch Laboratories, West Grove, PA) and the ECL Western Blotting Detection System (GE Healthcare Bio-Sciences, Piscataway, NJ).
9
Whole-mount Immunostaining of Phospho-ERK1/2 in Zebrafish
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-mount immunostaining was carried out as previously described [13] (link). Briefly, transgenic (flk:GFP) zebrafish embryos were treated with U0126 (MEK/ERK inhibitor, Cell Signaling Technology). The embryos were incubated with 40 µM U0126 for 14 h, fixed at 26 hpf, and stained with anti-phospho-ERK1/2 antibody (1∶500, Cell Signaling Technology). For fluorescent detection of the antibody, Alexa Fluor 568 anti-rabbit conjugate was used (1∶500, Molecular Probes, Grand Island, NY). All stained embryos were mounted with glycerol and photographed on an Olympus FV10i confocal microscope and their expression was analyzed by Image J software.
10
Immunoblot and qPCR Analysis of Protein and Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoblot analysis, cells were lysed by RIPA buffer and centrifuged to extract total proteins. The protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Scientific). A certain amount of protein was mixed with loading buffer (Beyotime Biotechnology), boiled for 15 min and subjected to SDS-PAGE followed by transferring to PVDF membranes. Blots were probed with primary antibodies, including anti-ATF6 antibody (Abcam, ab37149, 1:1000), anti-phospho-ERK1/2 antibody (Cell Signaling Technology, 4370, 1:1000), anti-ERK1/2 antibody (Cell Signaling Technology, 9102, 1:1000) and anti-FGFR3 antibody (Santa Cruz Biotechnology, sc-390,423, 1:1000). For qPCR, total RNA was extracted using MiniBEST Universal RNA Extraction Kit (TaKaRa). Reverse transcription was performed with PrimeScript RT Master Mix (TaKaRa). Synthesized cDNA was subjected to qPCR analysis using TB Green Premix Ex Taq II (TaKaRa). Primers used for qPCR to confirm Cre recombination were as follows: SLC26A2, 5′-AAG AGC AGC ATG ACC TCT CAC-3′ (forward) and 5’-CTG CCT CAA GTC AGT GCC T-3′ (reverse); GAPDH, 5′-AGG TCG GTG TGA ACG GAT TTG-3′ (forward) and 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′ (reverse).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!