Human/Mouse/Rat Phospho-ERK1(T202/Y204)/ERK2 (T185/Y187) Antibody

Detection of Human Phospho-ERK1 (T202/Y204) and ERK2 (T185/Y187) by Western Blot. Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or treated (+) with 200 nM PMA for for the indicated times. PVDF membrane was probed with 0.1 µg/mL of Rabbit Anti-Human/Mouse/Rat Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1018), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). Specific bands were detected for Phospho-ERK1 (T202/Y204) and ERK2 (T185/Y187) at approximately 42 and 44 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Phospho-ERK1/ERK2 in Jurkat Human Cell Line by Flow Cytometry. Jurkat human acute T cell leukemia cell line were untreated (yellow line open histogram) or treated with 200 ng/mL PMA for 15 minutes (filled histogram) and stained with Rabbit Anti-Human/Mouse/Rat Phospho-ERK1/ERK2 (ERK1 T202/Y204, ERK2 T185/Y187) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1018) or control antibody (Catalog # AB-105-C, blue line open histogram), followed by Fluorescein-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0112). To facilitate intracellular staining, cells were fixed with PFA and permeabilized with ice-cold methanol.

ERK1/ERK2 in Rat Brain. ERK1/ERK2 was detected in perfusion fixed frozen sections of rat brain (cortex) using 15 µg/mL Rabbit Anti-Human/Mouse/Rat Phospho-ERK1/ERK2 (ERK1 T202/Y204, ERK2 T185/Y187) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1018) overnight at 4 °C. Tissue was stained with the Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS005) and counterstained with hematoxylin (blue). View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.

Detection of Human Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) by Simple Western^TM. Simple Western lane view shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or treated (+) with PMA, loaded at 0.2 mg/mL. A specific band was detected for ERK1 (T202/Y204)/ERK2 (T185/Y187) at approximately 44 kDa (as indicated) using 5 µg/mL of Rabbit Anti-Human/Mouse/Rat Phospho-ERK1 (T202/Y204)/ERK2 (T185/Y187) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1018). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Mouse ERK1/ERK2 by Western Blot Biochemical analysis of ErbB signaling.Immunoblot analysis performed on P90 kidneys from Cdh16Cre::Tfebfs mice (A) and P90 Cdh16CreErt2::Tfebfs animals induced with tamoxifen at P14 (B) and at P30 (C), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:https://dx.doi.org/10.7554/eLife.17047.011 Image collected and cropped by CiteAb from the following publication (https://elifesciences.org/articles/17047), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse ERK1/ERK2 by Western Blot Activation of ErbB and WNT signaling pathways in kidneys from Cdh16Cre::Tfebfs mice.Transcriptional and biochemical analyses were performed on Cdh16Cre and Cdh16Cre::Tfebfs mice. (A,B) Tables show the relative increase of genes related to the ErbB (A) and WNT (B) pathways in the microarray analyses performed on kidneys from P0 Cdh16Cre::Tfebfs mice. Graphs show real-time PCR validations performed on kidneys from Cdh16Cre::Tfebfs mice at different stages (P0, P12, P30). Data are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice normalized versus wild-type mice. (C,D) Immunoblot analyses performed on (C) P30 kidney tissues and (D) primary kidney cells isolated from Cdh16Cre::Tfebfs mice to evaluate ErbB and WNT activation status. Each replicate is a distinct biological sample. ErbB signaling was assessed by looking at phosphoAKT (Ser473) to total AKT ratio, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK ratio; WNT signaling was assessed by quantifying beta -catenin and CCND1 (Cyclin D1) protein levels. Graphs represent the densitometry quantification of Western blot bands. Values are normalized to actin when not specified and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided, Student’s t test).DOI:https://dx.doi.org/10.7554/eLife.17047.00710.7554/eLife.17047.008Figure 3—source data 1.Complete list of 294 genes (represented by 361 probesets) significantly induced (FDR≤0.05) in the KSP_P0 microarray dataset (GSE62977).The genes are ranked by decreasing signed ratio (KSP_P0/CTL).DOI:https://dx.doi.org/10.7554/eLife.17047.00810.7554/eLife.17047.009Figure 3—source data 2.Complete list of 628 genes (represented by 729 probesets) significantly induced (FDR≤0.05) in the KSP_P14 microarray dataset (GSE63376).The genes are ranked by decreasing signed ratio (KSP_P14/CTL).DOI:https://dx.doi.org/10.7554/eLife.17047.009Complete list of 294 genes (represented by 361 probesets) significantly induced (FDR≤0.05) in the KSP_P0 microarray dataset (GSE62977).The genes are ranked by decreasing signed ratio (KSP_P0/CTL).DOI:https://dx.doi.org/10.7554/eLife.17047.008Complete list of 628 genes (represented by 729 probesets) significantly induced (FDR≤0.05) in the KSP_P14 microarray dataset (GSE63376).The genes are ranked by decreasing signed ratio (KSP_P14/CTL).DOI:https://dx.doi.org/10.7554/eLife.17047.009ErbB and WNT transcriptional profiles in Cdh16CreErt2::Tfebfs mice.Transcriptional analyses performed on Cdh16CreErt2::Tfebfs mice. (A,B) mRNA levels of previously validated genes belonging to the WNT (left graphs) and ErbB (right graphs) signaling pathways assessed in P90 Cdh16CreErt2::Tfebfs mice induced at (A) P14 and at (B) P30 with tamoxifen respectively. Data are shown as the average (± SEM) of at least Cdh16CreErt2::Tfebfs mice and values are normalized to the wild-type line. (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:https://dx.doi.org/10.7554/eLife.17047.010Biochemical analysis of ErbB signaling.Immunoblot analysis performed on P90 kidneys from Cdh16Cre::Tfebfs mice (A) and P90 Cdh16CreErt2::Tfebfs animals induced with tamoxifen at P14 (B) and at P30 (C), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:https://dx.doi.org/10.7554/eLife.17047.011 Image collected and cropped by CiteAb from the following publication (https://elifesciences.org/articles/17047), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse ERK1/ERK2 by Western Blot Biochemical analysis of ErbB signaling.Immunoblot analysis performed on P90 kidneys from Cdh16Cre::Tfebfs mice (A) and P90 Cdh16CreErt2::Tfebfs animals induced with tamoxifen at P14 (B) and at P30 (C), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:https://dx.doi.org/10.7554/eLife.17047.011 Image collected and cropped by CiteAb from the following publication (https://elifesciences.org/articles/17047), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse ERK1/ERK2 by Western Blot Biochemical analysis of ErbB signaling.Immunoblot analysis performed on P90 kidneys from Cdh16Cre::Tfebfs mice (A) and P90 Cdh16CreErt2::Tfebfs animals induced with tamoxifen at P14 (B) and at P30 (C), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:https://dx.doi.org/10.7554/eLife.17047.011 Image collected and cropped by CiteAb from the following publication (https://elifesciences.org/articles/17047), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human ERK1/ERK2 by Western Blot beta -blockers inhibit the proliferation of breast cancer cell lines(A) A dose curve of propranolol was administered to a panel of breast cancer cell lines and normal primary mammary epithelial cells (HMECS) and viability was assessed. (B) Immunofluorescent detection of Ki-67 protein expression in control or propranolol-treated (18 μM) SK-BR-3 breast cancer cells after 24 hours. Hoechst 33342 was used as a nuclear counterstain. (C) Heatmap depicting phospho-MAPK antibody array results testing the status of 24 kinases in SK-BR-3 cells subjected to 24 hours control or propranolol (18 μM). (red = upregulated; green = downregulated; black = no detectable expression). (D) Western blot confirmation of the antibody array results in SK-BR-3 cells subjected to 24 hours control or propranolol (18 μM). (D) Western blot analysis of cell lysates from SK-BR-3 cells subjected to 24 hours control or propranolol (18 μM), confirming the phosphorylation events identified in the antibody array. Image collected and cropped by CiteAb from the following publication (https://www.oncotarget.com/lookup/doi/10.18632/oncotarget.14119), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse ERK1/ERK2 by Western Blot Activation of ErbB and WNT signaling pathways in kidneys from Cdh16Cre::Tfebfs mice.Transcriptional and biochemical analyses were performed on Cdh16Cre and Cdh16Cre::Tfebfs mice. (A,B) Tables show the relative increase of genes related to the ErbB (A) and WNT (B) pathways in the microarray analyses performed on kidneys from P0 Cdh16Cre::Tfebfs mice. Graphs show real-time PCR validations performed on kidneys from Cdh16Cre::Tfebfs mice at different stages (P0, P12, P30). Data are shown as the average (± SEM) of at least three Cdh16Cre::Tfebfs mice normalized versus wild-type mice. (C,D) Immunoblot analyses performed on (C) P30 kidney tissues and (D) primary kidney cells isolated from Cdh16Cre::Tfebfs mice to evaluate ErbB and WNT activation status. Each replicate is a distinct biological sample. ErbB signaling was assessed by looking at phosphoAKT (Ser473) to total AKT ratio, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK ratio; WNT signaling was assessed by quantifying beta -catenin and CCND1 (Cyclin D1) protein levels. Graphs represent the densitometry quantification of Western blot bands. Values are normalized to actin when not specified and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided, Student’s t test).DOI:https://dx.doi.org/10.7554/eLife.17047.00710.7554/eLife.17047.008Figure 3—source data 1.Complete list of 294 genes (represented by 361 probesets) significantly induced (FDR≤0.05) in the KSP_P0 microarray dataset (GSE62977).The genes are ranked by decreasing signed ratio (KSP_P0/CTL).DOI:https://dx.doi.org/10.7554/eLife.17047.00810.7554/eLife.17047.009Figure 3—source data 2.Complete list of 628 genes (represented by 729 probesets) significantly induced (FDR≤0.05) in the KSP_P14 microarray dataset (GSE63376).The genes are ranked by decreasing signed ratio (KSP_P14/CTL).DOI:https://dx.doi.org/10.7554/eLife.17047.009Complete list of 294 genes (represented by 361 probesets) significantly induced (FDR≤0.05) in the KSP_P0 microarray dataset (GSE62977).The genes are ranked by decreasing signed ratio (KSP_P0/CTL).DOI:https://dx.doi.org/10.7554/eLife.17047.008Complete list of 628 genes (represented by 729 probesets) significantly induced (FDR≤0.05) in the KSP_P14 microarray dataset (GSE63376).The genes are ranked by decreasing signed ratio (KSP_P14/CTL).DOI:https://dx.doi.org/10.7554/eLife.17047.009ErbB and WNT transcriptional profiles in Cdh16CreErt2::Tfebfs mice.Transcriptional analyses performed on Cdh16CreErt2::Tfebfs mice. (A,B) mRNA levels of previously validated genes belonging to the WNT (left graphs) and ErbB (right graphs) signaling pathways assessed in P90 Cdh16CreErt2::Tfebfs mice induced at (A) P14 and at (B) P30 with tamoxifen respectively. Data are shown as the average (± SEM) of at least Cdh16CreErt2::Tfebfs mice and values are normalized to the wild-type line. (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:https://dx.doi.org/10.7554/eLife.17047.010Biochemical analysis of ErbB signaling.Immunoblot analysis performed on P90 kidneys from Cdh16Cre::Tfebfs mice (A) and P90 Cdh16CreErt2::Tfebfs animals induced with tamoxifen at P14 (B) and at P30 (C), respectively. Each replicate is a different biological sample. ErbB was analyzed by quantifying phosphoAKT (Ser473) to total AKT, and phosphoERK1 (T202/Y204)/ERK2(T185/Y187) to total ERK; graphs are the densitometry quantifications of Western blot bands normalized to wild-type line and are shown as an average (± SEM) (*p<0.05, **p<0.01, ***p<0.001, two-sided Student’s t test).DOI:https://dx.doi.org/10.7554/eLife.17047.011 Image collected and cropped by CiteAb from the following publication (https://elifesciences.org/articles/17047), licensed under a CC-BY license. Not internally tested by R&D Systems.