Total protein extracts (30 µg) were then electrophoresed in SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blots were blocked in tris-buffered saline (TBS) (50mM Tris-HCl, pH 7.5, 150mM NaCl and 0.05% Tween 20) with 5% dry milk and incubated with the following primary antibodies overnight at 4°C: anti-Arc/Arg3.1 antibody (1:800; Santa Cruz Biotechnology), anti-phospho-p44/42 ERK1/2 antibody (1:2000; Cell Signaling), anti-ERK1/2 antibody (1:1000; Cell Signaling), anti-phospho CREBSer133 antibody (1:1000; Cell Signaling), anti-CREB antibody (1:1000; Cell Signaling), anti-GluR1/2/3 antibody (1:1000; Millipore), and anti-β-Actin antibody (1:5000; Sigma). After washing three times for 5min in TBS/0.1% Tween-20, blots were then incubated with anti-rabbit or -mouse secondary antibody conjugated to horseradish peroxidase (1:2000; Zhongshan Biotechnology) and developed using the West Dura chemiluminescent substrate (Pierce Laboratories). Densitometry was determined based on band intensity, and relative protein expression was quantified by densitometry using the Total Lab 2.01 analysis system (Phoretix). To control for inconsistencies in loading, optical densities were normalized to β-actin protein expression. Data for treated animals were normalized to the average value of the naive controls.
Anti erk1 2 antibody
Manufactured by Cell Signaling Technology
Sourced in United States, China
The Anti-ERK1/2 antibody is a research-use only laboratory product that recognizes the extracellular signal-regulated kinases 1 and 2 (ERK1/2). ERK1/2 are serine/threonine protein kinases that play a key role in the regulation of cell proliferation, differentiation, and survival. This antibody can be used to detect and quantify ERK1/2 in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti phospho erk1 2 antibody, by Cell Signaling Technology (13 mentions)
Anti p erk1 2 antibody, by Cell Signaling Technology (12 mentions)
Anti akt antibody, by Cell Signaling Technology (10 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Anti β actin antibody, by Merck Group (4 mentions)
Anti gapdh antibody, by Cell Signaling Technology (4 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (4 mentions)
Anti p38 antibody, by Cell Signaling Technology (3 mentions)
Anti p p38 antibody, by Cell Signaling Technology (3 mentions)
Anti p38 mapk antibody, by Cell Signaling Technology (3 mentions)
Anti jnk antibody, by Cell Signaling Technology (3 mentions)
Anti phospho akt antibody, by Cell Signaling Technology (3 mentions)
Anti bcl 2 antibody, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti iκbα antibody, by Cell Signaling Technology (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Anti phospho crebser133 antibody, by Cell Signaling Technology (2 mentions)
Anti fak antibody, by Cell Signaling Technology (2 mentions)
Anti p fak antibody, by Cell Signaling Technology (2 mentions)
56 protocols using anti erk1 2 antibody
1
Western Blot Analysis of Synaptic Proteins
2
Western Blot Analysis of CXCR4 and Signaling
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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.
3
Regulating BBB Reporter via NS5A
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HeLa Tet-off cells (15 cm dish) were transfected with 15 μg of the parental BBB reporter plasmid or BBB plasmids containing 3′ UTR inserts along with 15 μg of the NS5A expression plasmid, mutated NS5A expression plasmids or mock expression plasmid, and 8 μg of the pTracerC GFP expression plasmid in 100–150 μl of Lipofectamine 2000. After transfection each 15 cm dish was split into five 10 cm dishes and 48 hours later cells were treated with 300 ng/ml of doxycycline. After 0, 1.5, 3, 4.5 or 6 h, total RNA was extracted using the RNeasy kit (Qiagen) following manufacturer's recommendations, and for each sample, 10 μg of RNA was analyzed by northern blot using beta-globin and GFP probes as described previously (33 (link)). To ensure expression of NS5A or mutated NS5A, protein was extracted from cells in duplicate plates 48 hours after transfection and analyzed by western blotting as described previously (34 (link)) using an anti-NS5A antibody (16 (link)) and an anti-ERK 1/2 antibody (Cell signaling).
4
Immunoblotting of Integrin, Akt, Erk, and FAK
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Immunoblotting was performed with rabbit anti‐ITGA3 antibody (1:250, HPA008572; SIGMA‐ALDRICH, St. Louis, MO, USA), anti‐Akt antibody (1:1000, #4691; Cell Signaling Technology, Danvers, MA, USA), anti‐p‐Akt antibody (1:1000, #4060; Cell Signaling Technology), anti‐Erk1/2 antibody (1:1000, #4695; Cell Signaling Technology) and anti‐p‐Erk1/2 antibody (1:2000, #4370; Cell Signaling Technology), anti‐FAK antibody (1:1000, #3285; Cell Signaling Technology) and anti‐p‐FAK antibody (1:1000, #8556; Cell Signaling Technology). Anti‐GAPDH antibodies (1:1000, ab8245; Abcam, Cambridge, UK) were used as an internal control. The procedures were performed as described in our previous studies.15 , 16 , 22
5
Western Blotting and IHC Analysis
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Western blotting and immunohistochemistry (IHC) methods were performed as described previously [22 (link)]. The IHC quantitation analysis was calculated by ImageJ software and statistically analyzed in three random fields. The antibodies used were as follows: anti-ISG20 antibody (Proteintech, Wuhan, China), anti-SEH1L antibody (Sigma, St. Louis, MO, USA), and anti-ERK1/2 antibody (Cell Signaling Technology, Danvers, MA, USA).
6
Quantitative Analysis of Retinal Protein Levels
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Retinal samples were prepared as described previously [16 (link)]. Twenty-five-microgram protein was subjected to SDS-PAGE and blotted to nitrocellulose membranes. Membranes were with anti-Nox4 antibody (Santa-Cruz), anti-p-VEGFR2, anti-p-ERK1/2 antibody, anti-ERK1/2 antibody, anti-p-P38 antibody, and anti-P38 MAPK antibody (Cell Signaling), followed by incubation with HRP-conjugated secondary antibodies (Vector Laboratories). The same membrane was reblotted with the anti-β-actin antibody (Sigma-Aldrich) as loading control.
7
Comprehensive Western Blot Analysis of Key Proteins
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Western blot analysis was performed as described previously [24 (link)]. The primary antibodies were as follows: anti-MUC15 (1: 1000 dilution, Immunoway, USA), anti β-actin (Abcam, USA), anti-AKT antibody (Abcam, USA), anti-p-AKT antibody (Abcam, USA), anti-Erk1/2 antibody (Cell Signaling Technology, USA), anti-pErk1/2 antibody (Cell Signaling Technology, USA), and anti-GAPDH (Santa Cruz, USA).
8
Thromboxane A2 Receptor Modulation in Neuroinflammation
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Thromboxane A2 receptors agonist U46619 and antagonist SQ29548 were purchased from Sigma Aldrich (St. Louis, MO, USA). MEK inhibitor U0126 was from Cell Signaling (Beverly, MA, USA). Anti-phospho-ERK antibody and anti-ERK1/2 antibody were from Cell Signaling (Beverly, MA, USA). Anti-CD11b antibody was from BD Biosciences (San Jose, CA, USA). TP antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Iba1 antibody was from BD Biosciences (San Jose, CA, USA). The Cell Counting kit-8 (CCK-8) was from KeyGen Biotech Co., Ltd. (Nanjing, China). NO assay kit was from Beyotime Institute of Biotechnology (Haimen, China). Adult male ICR mice and 24 h neonatal Sprague–Dawley (SD) rats were purchased from Shanghai SLAC Laboratory Animal Corporation (Shanghai, China). All animal procedures were carried out strictly within national guidelines and approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University.
9
Immunohistochemical Analysis of Apoptosis in Mouse Vagina
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Mice were sacrificed by intraperitoneal injection of pentobarbital sodium (Kyoritsuseiyaku Co., Tokyo, Japan) and then subjected to transcardiac perfusion of 4% paraformaldehyde. Each vagina excised from a mouse was fixed overnight in 4% paraformaldehyde solution. Each vagina was then embedded longitudinally in paraffin and cut into 4-µm serial sections. Sections were immunolabeled with anti-cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), anti-Akt (Cell Signaling Technology), or anti-Erk1/2 antibody (Cell Signaling Technology). TUNEL assays were performed basically as described previously [22] , using a fluorometric TUNEL assay system (Promega, Madison, WI) following the manufacturer’s protocols.
10
Western Blotting of Cerebral Cortex Proteins
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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).
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