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Erk1 2 k 23

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ERK1/2 (K-23) is a protein-specific antibody that recognizes the extracellular signal-regulated kinase 1/2 (ERK1/2) proteins. ERK1/2 are members of the mitogen-activated protein kinase (MAPK) family and play a crucial role in cellular processes such as proliferation, differentiation, and survival.

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3 protocols using erk1 2 k 23

1

Klotho Modulates Angiotensin II-Induced Vascular Remodeling

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Dulbecco’s modified Eagle’s medium (DMEM) was purchased from HyClone (HyClone, Logan, CT, USA). Fetal bovine serum (FBS) was purchased from ABGENT (San Diego, CA, USA). We purchased penicillin, streptomycin, and Angiotensin II from Sigma-Aldrich (St. Louis, MO, USA). The Cell Counting Kit-8 (CCK8) was purchased from Dojindo (Kumamoto, Japan). The rat recombinant Klotho protein was from Cloud-Clone Corporation (Houston, TX, USA). The antibodies used in this study were as follows: mouse monoclonal PCNA (PC10), rabbit polyclonal NF-κB p65 (C-20), rabbit polyclonal p-NF-κB p65 (Ser 536), rabbit polyclonal ERK1/2 (K-23), rabbit polyclonal p-ERK1/2 (Thr202/Tyr204), rabbit polyclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (FL-335) (Santa Cruz Biotechnology, CA, USA); and rabbit polyclonal SM22α (Cloud-Clone Corp., Wuhan, China). Anti-CDK2, anti-CDK4, anti-cyclin D, and anti-cyclin E antibodies were purchased from Wanleibio, Shenyang, China, and anti-Akt and anti-phospho-Akt (Ser473) antibodies were from Cell Signaling (MA, USA). The reactivity with rat antigens is included in all of these antibodies. Through ImageJ software (NIH, USA), the signal intensity was quantified.
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2

Immunoblotting and ChIP Assays for Cell Signaling Analysis

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The following antibodies were used at the indicated dilutions for immunoblotting: pJNK (#9251, 1:1000), p-p38 (#9211, 1:1000), pERK1/2 (#9101, 1:1000) and pSTAT3 antibodies (#9131, 1:1000), obtained from Cell Signaling; JNK (C-17, 1:1000), p38 (H-147, 1:1000), ERK1/2 (K-23, 1:1000), TRAF6 (H-274, 1:1000), anti-ubiquitin (P4D1, 1:1000), RAN (C-20, 1:1000), STAT3 (C-20, 1:1000), c-Myc (9E10, 1:5,000) and tubulin antibodies (10D8, 1:2000), obtained from Santa Cruz Biotech; and anti-K63 ubiquitin (HWA4C4, 1:750) and anti-Ubc13 antibodies (#371100, 1:1000), obtained from Enzo and Invitrogen, respectively. TRAF6 antibody (H-274) was also used for immunoprecipitation (1 g/sample). For ChIPs, the following antibodies were used at 2 g/sample: STAT3 (C-20) and c-Ets-1 antibodies (C-20), obtained from Santa Cruz Biotech; H3K4me3 (CS200580) and total H3 antibodies (04-928), obtained from Millipore; and Set1 antibody (A300-289A) from Bethyl Laboratories. For flow cytometry, fluorescently labeled antibodies to CD11b (M1/70, FITC-labeled), F4/80 (BM8, PerCP Cy5.5-labeled), M-CSFR (AFS98, APC-labeled) and RANK (R12-31, PE-labeled) from eBioscience were used at a 1:100 dilution. Recombinant murine RANKL, IL-6, IL-10 and IL-6 blocking antibody were purchased from R&D Systems.
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3

Immunoblotting and ChIP Assays for Cell Signaling Analysis

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The following antibodies were used at the indicated dilutions for immunoblotting: pJNK (#9251, 1:1000), p-p38 (#9211, 1:1000), pERK1/2 (#9101, 1:1000) and pSTAT3 antibodies (#9131, 1:1000), obtained from Cell Signaling; JNK (C-17, 1:1000), p38 (H-147, 1:1000), ERK1/2 (K-23, 1:1000), TRAF6 (H-274, 1:1000), anti-ubiquitin (P4D1, 1:1000), RAN (C-20, 1:1000), STAT3 (C-20, 1:1000), c-Myc (9E10, 1:5,000) and tubulin antibodies (10D8, 1:2000), obtained from Santa Cruz Biotech; and anti-K63 ubiquitin (HWA4C4, 1:750) and anti-Ubc13 antibodies (#371100, 1:1000), obtained from Enzo and Invitrogen, respectively. TRAF6 antibody (H-274) was also used for immunoprecipitation (1 g/sample). For ChIPs, the following antibodies were used at 2 g/sample: STAT3 (C-20) and c-Ets-1 antibodies (C-20), obtained from Santa Cruz Biotech; H3K4me3 (CS200580) and total H3 antibodies (04-928), obtained from Millipore; and Set1 antibody (A300-289A) from Bethyl Laboratories. For flow cytometry, fluorescently labeled antibodies to CD11b (M1/70, FITC-labeled), F4/80 (BM8, PerCP Cy5.5-labeled), M-CSFR (AFS98, APC-labeled) and RANK (R12-31, PE-labeled) from eBioscience were used at a 1:100 dilution. Recombinant murine RANKL, IL-6, IL-10 and IL-6 blocking antibody were purchased from R&D Systems.
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