Anti p erk1 2

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Germany, United Kingdom

Anti-p-ERK1/2 is a primary antibody reagent that specifically recognizes the phosphorylated forms of the extracellular signal-regulated kinases (ERK1/2). This antibody can be used to detect the activation state of these important signaling proteins.

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ERK1/2 (184 citations) P-ERK1/2 (130 citations) Anti-ERK1/2 (104 citations) Rabbit anti-p-ERK1/2 (4 citations) Anti-p-ERK1 (3 citations) Anti-p-Erk1/2 (Thr202/Tyr204) (sc-16982; (2 citations) Rabbit anti-p-ERK1/2 (sc-16982-R) (2 citations) Mouse anti- p-ERK1/2 (Tyr 204) (2 citations) Mouse monoclonal anti-p-ERK1/2 (2 citations) Anti-p-Erk1/2 (Thr202/Tyr204) (2 citations)

57 protocols using anti p erk1 2

Investigating SKA1 and Apoptosis Pathways

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TRIzol reagent and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA). The First Strand cDNA Synthesis kit and SYBR Premix Taq were from Takara (Dalian, Liaoning, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), bromodeoxyuridine (BrdU) and the anti-BrdU antibody were purchased from Sigma (St. Louis, MO, USA). DAPI, BCA protein assay and ECL Plus kits were obtained from Beyotime Institute of Biotechnology (Beijing, China). BD BioCoat Matrigel invasion chambers were purchased from BD Biosciences (San Jose, CA, USA).
The primary antibodies against human SKA1 and cleaved caspase-3 were obtained from Abcam (Cambridge, MA, USA). Anti-Bcl-2, anti-Bax, anti-p-ERK1/2, anti-ERK1/2, anti-p-Akt, anti-Akt, anti-p21, anti-cyclin D1 and anti-GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG secondary antibodies were purchased from Santa Cruz Biotechnology. Biotinylated- and Cy3-conjugated anti-rabbit secondary antibodies were purchased form Boster (Wuhan, Hubei, China).

SHEN L., YANG M., LIN Q., ZHANG Z., MIAO C, & ZHU B. (2016). SKA1 regulates the metastasis and cisplatin resistance of non-small cell lung cancer. Oncology Reports, 35(5), 2561-2568.

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Western Blot Analysis of Signaling Proteins

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Total cell-lysates were prepared, separated by SDS-PAGE, and submitted to Western blotting as described [62 (link),63 (link)]. Antibodies were used as follows: Anti-PD-L1 (cat. ab205921, Abcam, 1:1000 in Skim/TBST), anti-Hsp90 (cat. sc7947, 1:1000 in Skim/TBST), and anti-Gab1 (cat. sc133191, Santa Cruz, Heidelberg, Germany, 1:200 in Skim/TBST), anti-P-ERK1/2 (cat. 9101, 1:1000 in BSA/TBST), anti-P-AKT (cat. 9271, 1:2000 in BSA/TBST), anti-ERK1/2 (cat. 9102, 1:1000 in BSA/TBST), anti-AKT (cat. 9272, 1:2000 in BSA/TBST), anti-InsR (cat. 3025, 1:500 in BSA/TBST), anti-IGFR (cat. 14534, 1:500 in BSA/TBST), anti-rabbit HRP (cat 7074, 1:4000 in skim/TBST), and anti-mouse HRP (cat 7074, Cell Signaling Technology, Frankfurt, Germany, 1:4000 in skim/TBST), anti-αTubulin (cat. T5168, Sigma 1:10000, in Skim/TBST). Blots were analyzed with the ChemiDoc gel documentation system (Biorad, Munich, Germany). Relative band intensities were calculated by the QuantityOne software (Biorad) and the band densities were normalized to the corresponding housekeepers Hsp90 and tubulin or unphosphorylated Akt and ERK1/2, respectively.

Heckl S.M., Mau F., Senftleben A., Daunke T., Beckinger S., Abdullazade S., Schreiber S., Röcken C., Sebens S, & Schäfer H. (2021). Programmed Death-Ligand 1 (PD-L1) Expression Is Induced by Insulin in Pancreatic Ductal Adenocarcinoma Cells Pointing to Its Role in Immune Checkpoint Control. Medical Sciences, 9(3), 48.

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Protein Expression and Imaging Protocol

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Anti-PDIA1 (BD Bioscience, #610946, 1:1000), PDI Monoclonal Antibody RL90 (#MA3-019, 1:200) p-VEGFR2(1175) (Cell Signaling (CS), #3770, 1:1000), anti-VEGFR2 (CS#2479, 1:1000), anti-p-ERK1/2 (CS#9101, 1:1000), anti-ERK1/2 (CS#9102, 1:1000), anti-p-cSrc (CS#2101 1:1000), anti-cSrc (Santa Cruz # 5266, 1:1000), anti-PTP1B (D-4)(Santa Cruz, #133259, 1:1000), anti-Actin (Santa Cruz, #47778, 1:1000), anti-Rab5 (Santa Cruz #46692, 1:200), anti-Rab7 (B-3) (Santa Cruz# 376362, 1:200), anti CD31 (BD Biosciences# 550274, 1:200), anti-IsolectinB4 (Vector #B-1205, 1:200), anti-Flag (Sigma, #F7425, 1:1000) were used. Secondary antibodies, Goat Anti-Rabbit IgG–HRP conjugate (Bio Rad, #170-6515, 1:2000), Goat Anti-mouse IgG–HRP conjugate (Bio Rad, #170-6516, 1:2000), Alexa Fluor 568 goat anti Rat IgG (Invitrogen, # A-11077, 1:1000), Alexa Fluor 488 goat anti mouse IgG (Invitrogen, # A11001, 1:1000), Alexa Fluor-488-goat anti rabbit IgG (Invitrogen, #A11008), Alexa Fluor-546-goat anti mouse IgG (Invitrogen, #A11003), Alexa Fluor-546-goat anti rabbit IgG (Invitrogen, #A11010), Alexa Fluor-488-goat anti mouse IgG (Invitrogen, #A11001)were used.

Nagarkoti S., Kim Y.M., Ash D., Das A., Vitriol E., Read T.A., Youn S.W., Sudhahar V., McMenamin M., Hou Y., Boatwright H., Caldwell R., Essex D.W., Cho J., Fukai T, & Ushio-Fukai M. (2022). Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis. Angiogenesis, 26(1), 77-96.

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Western Blotting of Key Signaling Proteins

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Basic Western blotting methods were followed as previously described [4 (link)] using the following primary antibodies: ERα (Santa Cruz Biotechnology, Santa Cruz, CA), Flotillin-1 (BD Transduction Laboratories, Lexington, KY), anti-pERK1/2 (Santa Cruz Biotechnology), total ERK1/2 (Santa Cruz Biotechnology), anti-pAKT (Santa Cruz Biotechnology), and total Akt (Santa Cruz Biotechnology). Uncalibrated, optical density measurements were made using NIH Image 1.62 (National Institutes of Health, Bethesda, MD, USA) from scanned films.

Bains M, & Roberts J.L. (2015). Estrogen protects against dopamine neuron toxicity in primary mesencephalic cultures through an indirect P13K/Akt mediated astrocyte pathway. Neuroscience letters, 610, 79-85.

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Investigating VEGFR-Robo Signaling Pathway

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VEGF-C and Slit2N were purchased from ProSpec (East Brunswick, NJ) and
PreproTech, Inc. (Rocky Hill, NJ), respectively. Antibodies used include
anti-VEGFR-3, anti-p-Tyr, anti-GAPDH, and anti-p-ERK1/2 (Santa Cruz
Biotechnology, Santa Cruz, CA); anti-p-Akt (Ser-473), anti-VEGFR-2 (55B11),
anti-p-VEGFR-2 (Tyr1175), anti-ERK1/2 (9102), and anti-Akt (4685) (Cell
Signaling Technology, Beverly, MA); anti-PI3K p85 (Upstate Biotechnology,
Waltham, MA), anti-Robo1 (ab7279), and anti-Robo4 (ab10547) (Abcam Inc.,
Cambridge, MA).

Yu J., Zhang X., Kuzontkoski P.M., Jiang S., Zhu W., Li D.Y, & Groopman J.E. (2014). Slit2N and Robo4 regulate lymphangiogenesis through the VEGF-C/VEGFR-3 pathway. Cell Communication and Signaling : CCS, 12, 25.

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Protein Extraction and Western Blot Analysis

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Tissue protein was extracted with a protein extracting reagent (BioTeke Corporation, Beijing, China) according to the manufacturer's directions, while cell protein was extracted through RIPA buffer supplemented with 1% protease inhibitors (Roche, Basel, Switzerland). After centrifugation, the protein content was measured by a BCA Protein Assay Kit (Solarbio, Beijing, China). According to the standard procedures of western blot, total proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA), and then blocked with 5% skim milk in TBST. After being incubated with antibodies, the membranes were visualized with the ECL procedure (Millipore, USA) to get protein bands, which were analyzed by ImageJ software. The primary antibodies included anti-HMGN5, anti-Bcl-2, anti-Bax, anti-Cyclin D1, anti-p21, anti-MMP-2, anti-MMP-9, anti-AKT, anti-p-AKT, anti-ERK1/2, anti-p-ERK1/2 (Santa Cruz Biotechnology, Inc., USA), and anti-β-actin (WanleiBio, Shenyang, China). The secondary antibodies included HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (OriGene Technologies, USA).

Ma Q., Wang X., Wang H., Song W., Wang Q, & Wang J. (2020). HMGN5 Silencing Suppresses Cell Biological Progression via AKT/MAPK Pathway in Human Glioblastoma Cells. BioMed Research International, 2020, 8610271.

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Western Blot Analysis of Aorta and Macrophages

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Lysates of aorta or macrophages were prepared and then separated by 10% SDS‐PAGE. Briefly, cells were homogenized and lysed with RIPA lysis buffer. 40 μg protein per lane was separated by 12% SDS–PAGE and electroblotted onto a poly (vinylidene difluoride) membrane (GE Healthcare). Following that, non‐specific binding was blocked by incubating with 5% non‐fat milk in TBST buffer at room temperature for 1 hr. The transferred proteins were incubated overnight at 4°C with various primary antibodies in Tris‐buffered saline with Tween buffer (20 mM Tris‐HCl, 150 mM NaCl and 0.1% Tween 20) followed by incubation with horseradish peroxidase‐conjugated secondary antibodies for 1 hr. Primary antibodies including anti‐tenascin‐c (1:500; Santa Cruz Biotech), anti‐annexin II (1:500; Santa Cruz Biotech), anti‐Akt (1:500; Santa Cruz Biotech), anti‐p‐Akt (1:500; Santa Cruz Biotech), anti‐p65 (1:500; Santa Cruz Biotech), anti‐p‐p65 (1:600; Santa Cruz Biotech), anti‐ERK1/2 (1:800; Santa Cruz Biotech), anti‐p‐ERK1/2 (1:800; Santa Cruz Biotech), anti‐HIF‐1α (1:500; Santa Cruz Biotech) and anti‐HIF‐2α (1:600; Abcam, Cambridge, MA, USA) were employed. After extensive washes in Tris‐buffered saline with Tween buffer, the signals on the membrane were visualized by enhanced chemiluminescence (GE Healthcare).

Wang Z., Wei Q., Han L., Cao K., Lan T., Xu Z., Wang Y., Gao Y., Xue J., Shan F., Feng J, & Xie X. (2017). Tenascin‐c renders a proangiogenic phenotype in macrophage via annexin II. Journal of Cellular and Molecular Medicine, 22(1), 429-438.

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Western Blot Analysis of Protein Signaling

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Proteins were extracted from lung tissue and cell samples through homogenization in RIPA lysis and extraction buffer (ThermoFisher Scientific) according to the manufacturers’ instructions. After denature in SDS loading buffer, protein samples were separated by 8% or 10% SDS-PAGE and then transferred to PVDF membranes (Millipore). After block with 5% BSA diluted in TBST, the membranes were probed overnight at 4 °C with the following primary antibodies: anti-TRPM7 (Abcam, ab729), anti-β-actin (Santa Cruz, sc-81178), anti-cleaved caspase-3 (Cell Signaling Technology, 9661), anti-p-MEK 1/2 (Santa Cruz, sc-81503), anti-MEK 1/2 (Santa Cruz, sc-81504), anti-p-ERK 1/2 (Santa Cruz, sc-81492), anti-ERK 1/2 (Santa Cruz, sc-292838). After wash with TBST, the membranes were further incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). Protein bands were detected by chemiluminescence with the ECL detection reagent (Amersham Biosciences). The densitometric analysis of protein bands was performed by ImageJ software [48 (link)]. Results were normalized to those of β-actin.

Xing J., Wang M., Hong J., Gao Y., Liu Y., Gu H., Dong J, & Li L. (2019). TRPM7 channel inhibition exacerbates pulmonary arterial hypertension through MEK/ERK pathway. Aging (Albany NY), 11(12), 4050-4065.

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Isolation and Culture of Primary MEF Cells

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Primary MEF cells were isolated from embryos at E13.5 stages and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum as described (28 (link)). The cells were starved for 24 to 48 hours before FGF2 (104-02-50, ScienceCell) or IGF1 (291-G1-200, R&D Systems) treatment at 37°C. For gene deletions, MEF cells carrying homozygous flox alleles were infected with Ad5CMV-eGFP or Ad5CMVCre-eGFP (Gene Transfer Vector Core, University of Iowa, IA) in DMEM overnight at a multiplicity of infection of 500 plaque forming units per cell and cultured for 5 days. For inhibitor studies, cells were starved for 24 hours and treated for another 6 hours with PI3K inhibitors LY294002 (50 μM; #9901) and PX866 (1 μM; #13055), MEK inhibitor U0126 (50 μM; #9903), or mTOR inhibitor Torin (150 nM; #14385), all from Cell Signaling Technology. After growth factor stimulation, cells were washed in cold phosphate-buffered saline and harvested in ice-cold CelLytic reagent (C2978, Sigma-Aldrich) supplemented with protease inhibitor cocktail (78841, Thermo Fisher Scientific). Protein samples were denatured at 95°C for 5 min before loaded onto SDS–polyacrylamide gel electrophoresis gels. The antibodies used for Western blot were the same for immunohistochemistry, except anti-pERK1/2 (sc-7383, from Santa Cruz Biotechnology) and anti-ERK1/2 (#4695, Cell Signaling Technology).

Wang Q., Tao C., Hannan A., Yoon S., Min X., Peregrin J., Qu X., Li H., Yu H., Zhao J, & Zhang X. (2021). Lacrimal gland budding requires PI3K-dependent suppression of EGF signaling. Science Advances, 7(27), eabf1068.

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Western Blot Analysis of Cell Signaling

Cited 1 time

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Western blots were performed as described [41 (link)]. Primary antibody: anti-PPAR-α (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany, sc-398394), anti-TRL4 (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany, sc-293072), anti-pERK 1/2 (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany, sc-7383), anti-NLRP3 (1:500, Santa Cruz Biotechnology, Heidelberg, Germany, sc-134306), anti-transforming growth factor-beta3 (TGF-β3), (1:1000, Santa Cruz Biotechnology, Heidelberg, Germany, sc-166833), anti-claudin-11 (1:100, Santa Cruz Bio-technology, Heidelberg, Germany, sc-271232), or anti-occludin (1:100, Santa Cruz Biotechnology, Heidelberg, Germany, sc-133255) [44 (link),45 (link)].

Antonuccio P., Marini H.R., Micali A., Romeo C., Granese R., Retto A., Martino A., Benvenga S., Cuzzocrea S., Impellizzeri D., Di Paola R., Fusco R., Cervellione R.M, & Minutoli L. (2021). The Nutraceutical N-Palmitoylethanolamide (PEA) Reveals Widespread Molecular Effects Unmasking New Therapeutic Targets in Murine Varicocele. Nutrients, 13(3), 734.

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