Rabbit anti phosphorylated erk1 2

Manufactured by Cell Signaling Technology

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Rabbit anti-phosphorylated ERK1/2 is an antibody that recognizes the phosphorylated forms of extracellular signal-regulated kinase 1 and 2 (ERK1/2). ERK1/2 are important signaling proteins that play a role in cellular processes such as proliferation, differentiation, and survival. This antibody can be used to detect the activated, phosphorylated state of ERK1/2 in various biological samples.

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ERK1/2 (1487 citations) Anti-ERK1/2 (692 citations) Rabbit anti-ERK1/2 (92 citations) Phosphorylated ERK1/2 (76 citations) Anti-phosphorylated ERK1/2 (23 citations)

7 protocols using rabbit anti phosphorylated erk1 2

Immunohistochemical Analysis of AMD Patient Specimens

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AMD patient specimens were obtained in our clinic by enucleation due to suspected melanoma from an 82-year-old male with massive subretinal and vitreous hemorrhage secondary to CNV. This study was approved by the Ethics Committee of Hokkaido University Hospital, and written informed consent was obtained from the patient after an explanation of the purpose and consequence of this study. The enucleated globe was fixed with 4% paraformaldehyde and embedded with paraffin. Sections were deparaffinized and hydrated through exposure with xylene and graded alcohols followed by water. As a pretreatment, microwave-based antigen retrieval was performed in 10 mM citrate buffer (pH 6). These slides were incubated with the following primary antibodies: mouse anti-(P)RR (1:50),¹⁸ (link) rabbit anti-(P)RR (1:50; Sigma-Aldrich), rabbit anti-CD34 (1:100) and mouse anti-RPE65 (1:100; Abcam), and rabbit anti-phosphorylated ERK1/2 (1:100; Cell Signaling Technology) antibodies.

Liu Y., Kanda A., Wu D., Ishizuka E.T., Kase S., Noda K., Ichihara A, & Ishida S. (2019). Suppression of Choroidal Neovascularization and Fibrosis by a Novel RNAi Therapeutic Agent against (Pro)renin Receptor. Molecular Therapy. Nucleic Acids, 17, 113-125.

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Antibody Validation for IHC, IF, and Western Blotting

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Antibodies to the following were used for IHC, Immunofluorecence(IF) and western blotting: rat anti-mouse Kit (clone ACK4, Cedarlane, CL8936ap, 1:100 for IF), rabbit anti-KIT (Cell signaling, #3074, 1:100 for IHC), rabbit anti-Ki67 (Abcam, ab16667, 1:400 for IHC), rabbit anti-phosphorylated ERK1/2 (Cell signaling, #4377, 1:5000 for western blotting, 1:200 for IF), rabbit anti-MEK1/2 (Cell signaling, #2338, 1:2000 for western blotting), rabbit anti-phosphorylated p90RSK (Cell signaling, #12032, 1:1000 for western blotting), rabbit anti-ETV1 (Abcam, #ab81086, 1:500 for western blotting), HRP-conjugated anti-actin (Abcam, ab49900, 1:5000 for western blotting).
The dabrafenib was purchased from Active Biochem.

Ran L., Murphy D., Sher J., Cao Z., Wang S., Walczak E., Guan Y., Xie Y., Shukla S., Zhan Y., Antonescu C.R., Chen Y, & Chi P. (2017). ETV1-positive cells give rise to BRAFV600E mutant gastrointestinal stromal tumors. Cancer research, 77(14), 3758-3765.

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Western Blot Analysis of Signaling Proteins

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The tissues and cells were lysed in SDS buffer. After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins (20 μg) were resolved by SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA) by electroblotting. Membranes were incubated with the following primary antibodies: rabbit anti-phosphorylated ERK1/2 (1:1,000), rabbit anti-ERK1/2 (1:1,000), rabbit anti-phosphorylated SMAD2 (1:1,000), rabbit anti-SMAD2 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-(P)RR antibody (1:1,000; Sigma-Aldrich), and mouse anti-GAPDH (1:2,000; Thermo Fisher Scientific). Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgGs (1:4,000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used as secondary antibodies for chemoluminescence detection. Signals were obtained by enhanced chemoluminescence (Western Lightning Ultra; Perkin Elmer, Waltham, MA, USA). The bands were analyzed by densitometry using ImageJ software (NIH, Bethesda, MD, USA).

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Immunoblotting Analysis of Protein Signaling

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Cell extracts were lysed in SDS buffer, a protease inhibitor cocktail (Promega) and a phosphatase inhibitor cocktail (FUJIFILM Wako Pure Chemical Corporation). After quantifying protein concentrations using BCA reagent (Thermo Fisher Scientific), proteins were resolved by SDS‐PAGE (polyacrylamide gel electrophoresis) and transferred to nitrocellulose membrane by electroblotting. Membranes were blocked in TBS containing 5% skim milk and probed with the following primary antibodies: goat anti‐galecint‐1 (1:1000, R&D systems), mouse anti‐TSC22D3 (1:500), mouse anti‐ubiquitin (1:500, Santa Cruz Biotechnology), rabbit anti‐HIF‐1α (1:1000), rabbit anti‐phosphorylated AKT (1:2000), rabbit anti‐AKT (1:2000), rabbit anti‐phosphorylated ERK1/2 (1:2000), rabbit anti‐ERK1/2 (1:2000, Cell signaling technology) and rabbit anti‐β‐actin (1:4000, Medical & Biological Laboratories) antibodies. Horseradish peroxidase‐conjugated anti‐goat, anti‐mouse and anti‐rabbit IgGs (Jackson ImmunoResearch Laboratories) were used as a secondary antibody for chemoluminescence detection. Signals were visualized using a SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).

Kanda A., Hirose I., Noda K., Murata M, & Ishida S. (2020). Glucocorticoid‐transactivated TSC22D3 attenuates hypoxia‐ and diabetes‐induced Müller glial galectin‐1 expression via HIF‐1α destabilization. Journal of Cellular and Molecular Medicine, 24(8), 4589-4599.

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Western Blot Analysis of hPDLSCs

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The protein from shear stress-induced hPDLSCs was extracted by RIPA buffer containing protease inhibitor buffer (cat. No. P8340, Sigma-Aldrich). The concentration of lysate proteins was measured with a BCA protein assay (PierceTM BCA detection reagent, cat No. 23228 and 23224, Thermo Scientific). Lysate proteins were separated on 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was placed in a solution containing a monoclonal antibody to rabbit-anti actin (cat. No. A2066, Sigma-Aldrich), rabbit anti-TGF-

β

1 (cat No. ab92486, Abcam), rabbit anti-ERK1/2 (T202/Y204, Cell Signaling) or rabbit anti-phosphorylated ERK1/2 (cat. No. 137F5, Cell Signaling Technology) antibodies. Then, the membranes were developed with the horseradish peroxidase-linked antibody (cat No. 7074S, Cell signaling Technology). The membranes were visualised and exposed to chemiluminescence (SuperSignal^® West Pico Chemiluminescent Substrate, cat No. 34577, Thermo Scientific) and an image analyser (GE Healthcare, Pittsburgh, PA, USA), respectively. The band density was measured using ImageJ software. The band density was normalised to band density of actin and to the control.

Suwittayarak R., Klincumhom N., Ngaokrajang U., Namangkalakul W., Ferreira J.N., Pavasant P, & Osathanon T. (2022). Shear Stress Enhances the Paracrine-Mediated Immunoregulatory Function of Human Periodontal Ligament Stem Cells via the ERK Signalling Pathway. International Journal of Molecular Sciences, 23(13), 7119.

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Bladder Cancer Cell Lines and ERK Pathway Inhibition

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The RT-4, BI-87,253 J, SV-HUC-1, T24 and J82 human bladder tumor cell lines were purchased from China Academia Sinica Cell Repository (Shanghai, China; www.cellbank.org.cn). T24, RT-4, and 253 J cells were cultured in RPMI 1640 (cat. no. 22400-089; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.). J82 cells were maintained in Opti-Minimal Essential Medium^® I (cat. no. 51985-042; Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.). SV-HUC-1 cells were cultured in F12K medium (cat. no. N3520; Sigma-Aldrich; Merck Millipore) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) All cells were cultured in a humidified incubator at 37°C and 5% CO₂. The following antibodies were used: Anti-B23 (cat. no. 10306-1-AP; Proteintech Group, Inc., Rosemont, USA), mouse anti-GAPDH (cat. no. sc-32233; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit anti-ERK (cat. no. 4372S; Cell Signaling Technology, Inc., Danvers, MA, USA) and rabbit anti-phosphorylated (p)-ERK1/2 (cat. no. 4370S; Cell Signaling Technology, Inc.). U0126 (cat. no. 9903; Cell Signaling Technology, Inc.) was selected as the mitogen activated protein kinase (MAPK)/ERK inhibitor and the cells were treated with 2 µM U0126 for 1, 2, 3 days at 37°C.

Wang H., Yuan G., Zhao B., Zhao Y, & Qiu Y. (2016). High expression of B23 is associated with tumorigenesis and poor prognosis in bladder urothelial carcinoma. Molecular Medicine Reports, 15(2), 743-749.

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Western Blot Analysis of Protein Markers

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The extracts from AML12 cells and liver tissues were subjected to electrophoresis separation. Then, the extracts on the gel were transferred to the polyvinylidene fluoride membrane (1620177, BIO-RAD) which was blocked with 5% skim milk or 5% BSA for 1 h at ambient temperature. The membrane was incubated with primary antibodies of rabbit anti-β-actin (4970, 1:5000, Cell Signaling Technology), rabbit anti-AKR1B10 (OAGA00776, 1:1000, Aviva Systems Biology), rabbit anti-Caspase-3 (ab184787, 1:1000, Abcam), rabbit anti-Cleaved Caspase-3 (9661, 1:1000, Cell Signaling Technology), rabbit anti-phosphorylated (p)-Erk1/2 (4370, 1:1000, Cell Signaling Technology), and rabbit anti-Erk1/2 (4695, 1:1000, Cell Signaling Technology) at 4 °C overnight. The next day, the membrane was incubated with horseradish peroxide labeled goat anti-rabbit IgG (ab6721, 1:5000, Abcam) secondary antibody for l h at ambient temperature. The membrane was immersed in the enhanced chemiluminescence reaction solution (1705062, BIO-RAD) and imaged utilizing the Image Quant LAS 4000 C Gel Imager (GE, USA).

Wu Y., Qi Y., Bai Y., Zhang H., Zhu W., Zhou S, & Zhang Y. (2022). LncRNA 1700020I14Rik promotes AKR1B10 expression and activates Erk pathway to induce hepatocyte damage in alcoholic hepatitis. Cell Death Discovery, 8, 374.

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