E2HSA (Patent no. CN101525386A) was provided by Zhejiang Huayang Pharma Inc. (China) as freeze-dried powder. Exendin-4 (exenatide) was a product of Eli Lilly and Company (USA). Lipofectamine 2000 was obtained from Invitrogen (USA). Rat anti-insulin antibody was purchased from R&D Inc. (USA). Rabbit anti-glucagon antibody, rabbit anti-FoxO1 antibody, rabbit anti-phospho-FoxO1 antibody, rabbit anti-BAD antibody, rabbit anti-phospho-BAD antibody, rabbit anti-Bim antibody, rabbit anti-Bcl-2 antibody, rabbit anti-Bcl-XL antibody, and rabbit anti-Phospho-Erk1/2 antibody were all purchased from Cell Signaling Technology Inc. (USA). The in situ cell death detection kit was a product of Roche Inc. (USA).
Rabbit anti phospho erk1 2 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit 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. It is designed for use in various immunoassay techniques, such as Western blotting and immunohistochemistry, to analyze the activation state of the ERK1/2 signaling pathway.
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Lab products found in correlation
Rabbit anti erk1 2 antibody, by Cell Signaling Technology (4 mentions)
Rabbit anti phospho p38 mapk antibody, by Cell Signaling Technology (3 mentions)
Mouse anti erk1 2 antibody, by Cell Signaling Technology (2 mentions)
Rabbit anti sapk jnk antibody, by Cell Signaling Technology (2 mentions)
Mouse anti phospho sapk jnk antibody, by Cell Signaling Technology (2 mentions)
Rabbit anti p38 mapk antibody, by Cell Signaling Technology (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rabbit anti foxo1 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti glucagon antibody, by Cell Signaling Technology (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Rabbit anti bim antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti bcl 2 antibody, by Cell Signaling Technology (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Skov3, by American Type Culture Collection (1 mentions)
Rabbit anti caspase 3 antibody, by Cell Signaling Technology (1 mentions)
Mouse anti β actin antibody, by Cell Signaling Technology (1 mentions)
Mouse anti akt antibody, by Cell Signaling Technology (1 mentions)
Mtt reagent, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Rabbit anti phospho jnk antibody, by Cell Signaling Technology (1 mentions)
11 protocols using rabbit anti phospho erk1 2 antibody
1
Exendin-4 Mediated Pancreatic Cell Protection
2
Investigating SKOV-3 Ovarian Cancer Cells
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Human ovarian adenocarcinoma cells (SKOV-3) were purchased from American Type Culture Collection (Manassas, VA, USA). RES was purchased from MilliporeSigma (Burlington, MA, USA). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from MilliporeSigma. Guava® Cell Cycle Reagent was purchased from Luminex Corporate (Austin, TX, USA). Annexin V was purchased from ImmunoTools (Friesoythe, Niedersachsen, Germany). Propidium iodide was purchased from MilliporeSigma. Rabbit anti-phospho p38 antibody, rabbit anti p38 antibody, rabbit anti-phospho ERK1/2 antibody, mouse anti-ERK1/2 antibody, rabbit anti-phospho JNK antibody, rabbit anti-JNK antibody, rabbit anti-phospho AKT (Ser473) antibody, mouse anti-AKT antibody, rabbit anti-cyclin A2 antibody, rabbit anti-cyclin B1 antibody, rabbit anti-cyclin E1 antibody, rabbit anti-caspase-9 antibody, rabbit anti-caspase-3 antibody, rabbit anti-PARP antibody, mouse anti-β-actin antibody, and DAPI (4, 6-diamidino-2-phenylindole, dihydrochloride) were purchased from Cell Signaling Technology (Danvers, MA, USA). Goat anti-mouse IgG-IRDye®800CW and goat anti-rabbit IgG-IRDye®680RT were purchased from Li-COR Biosciences (Lincoln, NE, USA). Goat anti-rabbit conjugated with Alexa488 and Goat anti-mouse conjugated with Alexa594 were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
3
Western Blot Analysis of Brain Tissues
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The samples of brain tissues were homogenized in RIPA lysis buffer with protease or phosphatase inhibitor (Roche, Basel, Switzerland) and then centrifuged (12000 rpm, 15 min, 4°C) to obtain supernatants. The total protein concentration of the supernatants was determined using the BCA assay kit (Pierce). Samples were loaded and separated on the SDS–PAGE gel (40 μg per well). The protein was transferred to the PVDF membrane (Millipore, MA, United States), which was incubated overnight with primary antibodies at 4°C and then probed with the corresponding secondary antibody. The primary antibodies were as follows: rabbit anti-AT1R antibody (1:800 dilution, Abcam, MA, United States) (Czikora et al., 2015 (link); Wang et al., 2018 (link)), rabbit anti-SAPK/JNK antibody, mouse anti-phospho-SAPK/JNK antibody, mouse anti-ERK1/2 antibody, rabbit anti-phospho-ERK1/2 antibody, rabbit anti-p38MAPK antibody, rabbit anti-phospho-p38MAPK antibody and rabbit anti-β-tublin antibody (1:1000 dilution, Cell Signaling Technology, Danvers, MA, United States). The membranes were detected by an ECL-Plus detection kit (Tiangen, Beijing, China), and scanned using Image Quant LAS 4000 (GE Healthcare Life Sciences, CT, United States). The images were quantified using the ImageJ densitometry system.
4
Immunoblotting Analysis of Insulin Receptor Signaling
5
NMU Signaling Pathway Analysis
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DMEM medium, RPMI 1640 medium, penicillin, streptomycin, glutamine and zeocin were purchased from Invitrogen (Carlsbad, CA). Human NMU peptide was obtained from Phoenix Pharmaceuticals (Burlingame, CA). The iodination kit was from Pierce Thermo Scientific (Rockford, IL). Rabbit anti-phospho-ERK1/2 antibody and rabbit anti-ERK2 antibody were purchased from Cell Signaling Technology (Danvers, MA) and Santa Cruz Biotechnology (St. Cruz, CA), respectively. Mouse anti-FLAG antibody, rabbit FITC-conjugated anti-mouse IgG secondary antibody and other chemicals unless noted otherwise were purchased from Sigma (St. Louis, MO).
6
Embryoid Body Formation from ESCs
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To mimic embryonic development in a three-dimensional manner, we used a hanging drop method to differentiate ESCs into EBs. Briefly, ESC colonies were dissociated into single cells and suspended in ESC medium without LIF. To induce formation of EBs, 1000 cells were plated in a 20 µL drop hanging on the lid of culture dish and cultured for 3 days. EBs were collected at day 3 and transferred to ultra-low attachment 6-well plate (Corning) containing fresh ESC medium without LIF. For further suspension culture, medium was refreshed at day 5. EBs were harvested at day 3, 5, and 7 for analysis. For Western blot analysis, total proteins were prepared from ESCs and EBs at day 3, 5, and 7 to detect expressions of HO-1, Oct4, brachyury (R&D, AF2085), SM α-actin (Sigma, A5228), SM22α (Abcam, ab155272), or Smad2 (Cell signaling, #3102). The blots were subsequently probed with α-tubulin antibody (GeneTex, GTX112141) to verify loading. To assess Erk1/2 phosphorylation, blots were probed with a rabbit anti-phospho-Erk1/2 antibody (Cell Signaling Technology, #9101) and α-tubulin antibody for loading.
7
Immunohistochemistry of Phosphorylated Kinases
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The procedure for immunohistochemistry was described previously (Peng et al., 2009 (link)). One section from every six serial sections was picked up, with a total of five sections in each animal. Tissue sections were deparaffinized, rehydrated by graded ethanol series, pre-incubated with 5% BSA for an hour, and then incubated with mouse anti-phospho-SAPK/JNK antibody (1:50 dilution, Cell Signaling Technology, Danvers, MA, United States), rabbit anti-phospho-ERK1/2 antibody (1:200 dilution, Cell Signaling Technology, Danvers, MA, United States), or rabbit anti-phospho-p38MAPK antibody (1:400 dilution, Cell Signaling Technology, Danvers, MA, United States) at 4°C overnight. The sections were washed three times with PBS and then incubated with corresponding secondary antibodies. The color reaction was carried out with HRP-linked polymer detection system.
8
Recombinant IL-25 and Angiogenesis Pathway
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Recombinant mouse IL-25 (rmIL-25, Cat#: 587306) was brought from Biolegend, recombinant human IL-25 (rhIL-25, Cat#: 8134-IL) was purchased from R&D systems and prepared following instructions. Rabbit anti-CD31 antibody (Cat#: GB11063-2) was obtained from Servicebio. Goat anti-IL-17RB antibody (Cat#: AF1040) was obtained from R&D systems. Rabbit anti-VEGF antibody (Cat#: ab52917) and rabbit anti-β-catenin antibody (Cat#: ab32572) was from Abcam. Mouse anti-GAPDH antibody (Cat#: 60004-1-Ig) and rabbit anti-IL-17RB antibody (Cat#: 20673-1-AP) were obtained from Proteintech. Rabbit anti-phospho-Akt antibody (Cat#: 4060T), rabbit anti-Akt antibody (Cat#: 4091T), rabbit anti-phospho-ERK 1/2 antibody (Cat#: 4370S) and rabbit anti-ERK 1/2 antibody (Cat#: 4695T) were purchased from Cell Signaling Technology (CST). Glucose and streptozotocin (STZ, Cat#: S0130) were purchased from Sigma-Aldrich; and pLenti-CMV-IL-25-GFP-puro lentiviral plasmid (Lenti-IL-25-GFP, Cat#: PPL02165-4a) was obtained from Public Protein/Plasmid Library (PPL). The lentiviral packaging plasmid pMD2.G (Cat#: 12259) and psPAX2 (Cat#: 12260) were obtained from AddGene. Basement membrane matrix (Cat#: 354234) was obtained from Corning.
9
Western Blot Analysis of Patched and Erk1/2
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Total RIPA extracts from cells or tumor homogenates were prepared. Protein concentrations were determined by the DC Protein Assay (Bio-Rad, Marnes-la-Coquette, France). Samples (50 to 80 µg) were separated on SDS-PAGE and transferred to nitrocellulose membranes (Amersham, Bath, UK) using standard techniques. After 1 h at room temperature in blocking buffer (20 mmol/L Tris-HCl pH 7.5, 45 mmol/L NaCl, 0.1% Tween-20, and 5% non-fat milk), nitrocellulose membranes were incubated overnight at 4 °C with rabbit anti-Patched antibody (Abcam ab53715; 1/1000), rabbit anti-Phospho-Erk1/2 antibody (Cell Signaling Technology (Leiden, The Netherlands); 1/1000), rabbit anti-Erk1/2 antibody (Cell Signaling Technology; 1/1000), or mouse anti-β-tubulin antibody (Sigma; 1/1000). After 3 washes, membranes were incubated for 45 min with anti-rabbit (1:2000) or anti-mouse (1:5000) immunoglobulin coupled to horseradish peroxidase (Dako-Agilent, Santa Clara, CA, USA). Detection was carried out with an ECL Prime Western blotting detection reagent (Amersham) on a Fusion FX imager (Vilber Lourmat, Collegien, France), and analyses were performed using ImageJ software.
10
Western Blot Analysis of Signaling Pathways
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The antibodies used were rabbit anti-p44/42 MAPK (Erk1/2) antibody #9102 (Cell Signaling Technology, Beverly, MA), rabbit anti-phospho-Erk1/2 antibody #9101 (Cell Signaling Technology), rabbit anti-phospho-p38 MAPK antibody #4511 (Cell Signaling Technology), rabbit anti-p38 MAPK antibody #8690 (Cell Signaling Technology), rabbit anti-phospho-SAPK/JNK antibody #4668 (Cell Signaling Technology), rabbit anti-SAPK/JNK antibody #9258 (Cell Signaling Technology), mouse anti-phospho-IκBα #9246 (Cell Signaling Technology), mouse anti-IκBα antibody #4814 (Cell Signaling Technology), rabbit anti-LTβR, N-Terminal antibody #SAB4501788 (Sigma-Aldrich), goat anti-LTβR antibody #L5412 (Sigma-Aldrich), normal goat IgG control #AB-108-C (R&D systems), anti-mouse-IgG HRP-linked antibody #7076 (Cell Signaling Technology), and anti-rabbit IgG-HRP-linked antibody #7074 (Cell Signaling Technology).
Equal protein loading was confirmed by probing the blot with mouse anti-α-tubulin (Sigma-Aldrich) antibody.
Equal protein loading was confirmed by probing the blot with mouse anti-α-tubulin (Sigma-Aldrich) antibody.
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