Human/Mouse/Rat Phospho-Akt (S473) Pan Specific Antibody

Detection of Mouse and Human Phospho-Akt (S473) Pan Specific by Western Blot. Western blot shows lysates of NIH-3T3 mouse embryonic fibroblast cell line untreated (-) or treated (+) with 100 ng/mL Human PDGF (120-HD) for 15 minutes and MCF-7 human breast cancer cell line untreated or treated with 100 ng/mL Recombinant Human IGF-1 (291-G1) for 15 minutes. PVDF membrane was probed with 0.5 µg/mL of Rabbit Anti-Human/Mouse/Rat Phospho-Akt (S473) Pan Specific Antigen Affinity-purified Polyclonal Antibody (Catalog # AF887), followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (HAF008). A specific band was detected for Phospho-Akt (S473) Pan Specific at approximately 60 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Akt in Human Breast Cancer Tissue. Akt phosphorylated at S473 was detected in immersion fixed paraffin-embedded sections of human breast cancer tissue using Rabbit Anti-Human/Mouse/Rat Phospho-Akt (S473) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF887) at 10 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (brown; CTS005) and counterstained with hematoxylin (blue). Lower panel shows a lack of labeling if primary antibodies are omitted and tissue is stained only with secondary antibody followed by incubation with detection reagents. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.

Detection of Phospho-Akt (S473) in IGF-1-treated MCF‑7 cells by Flow Cytometry. Serum-starved MCF-7 human breast cancer cells were unstimulated (blue open histogram) or activated with 100 ng/mL Recombinant human IGF-1 (Catalog # 291-G1, dark orange filled histogram) for 15 minutes, then stained with Rabbit Anti-Human/Mouse/Rat Phospho-Akt (S473) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF887) followed by PE-conjugated Anti-Rabbit IgG Secondary Antibody (F0110). To facilitate intracellular staining, cells were fixed with formaldehyde (FC004) and permeabilized with Methanol.

Detection of Human Phospho-Akt (S473) by Simple Western^TM. Simple Western lane view shows lysates of MCF-7 human breast cancer cell line and A549 human lung carcinoma cell line untreated (-) or treated (+) with 100 ng/mL Recombinant Human IGF-I (291-G1) for 15 minutes, loaded at 0.2 mg/mL. A specific band was detected for Phospho-Akt (S473) at approximately 60 kDa (as indicated) using 5 µg/mL of Rabbit Anti-Human/Mouse/Rat Phospho-Akt (S473) Pan Specific Antigen Affinity-purified Polyclonal Antibody (Catalog # AF887). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.

Detection of Human AKT by Western Blot Phospho-proteome profiling results of LX-2 cells co-cultured with C3A–CYP2E1 cells with or without DDC treatment and detection of the effect of DDC on intracellular kinases in LX-2 cells of co-cultures200 μg of total cell lysates from LX-2 cells co-cultured with C3A-2E1 cells with or without 100 μM DDC for 1 h were incubated with membranes of the human phospho-MAPK Array Kit according to the manufacturer's instructions. Phospho MAPK Array data were developed on X-ray films following exposure to chemiluminescent reagents. 20 μg aliquots of total cell lysates from LX-2 cells were subjected to Western blotting analysis. (A) Template showing the location of MAPK antibodies spotted onto the human phospho-MAPK Array Kit. (B) The activation status of ERK1/2 in LX-2 cells co-cultured with C3A-2E1 cells after DDC treatment. (C) The activation status of p38 in LX-2 cells co-cultured with C3A-2E1 cells after DDC treatment. (D) The activation status of Akt in LX-2 cells co-cultured with C3A-2E1 cells after DDC treatment. (E) The activation status of ERK1/2, p38 and Akt in LX-2 cells co-cultured with C3A cells after DDC treatment. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/23577625), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human AKT by Western Blot Phospho-proteome profiling results of LX-2 cells co-cultured with C3A–CYP2E1 cells with or without DDC treatment and detection of the effect of DDC on intracellular kinases in LX-2 cells of co-cultures200 μg of total cell lysates from LX-2 cells co-cultured with C3A-2E1 cells with or without 100 μM DDC for 1 h were incubated with membranes of the human phospho-MAPK Array Kit according to the manufacturer's instructions. Phospho MAPK Array data were developed on X-ray films following exposure to chemiluminescent reagents. 20 μg aliquots of total cell lysates from LX-2 cells were subjected to Western blotting analysis. (A) Template showing the location of MAPK antibodies spotted onto the human phospho-MAPK Array Kit. (B) The activation status of ERK1/2 in LX-2 cells co-cultured with C3A-2E1 cells after DDC treatment. (C) The activation status of p38 in LX-2 cells co-cultured with C3A-2E1 cells after DDC treatment. (D) The activation status of Akt in LX-2 cells co-cultured with C3A-2E1 cells after DDC treatment. (E) The activation status of ERK1/2, p38 and Akt in LX-2 cells co-cultured with C3A cells after DDC treatment. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/23577625), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human AKT by Western Blot DDC up-regulates MMP-1 through ERK1/2 and Akt activation(A) Co-cultures were treated with 100 μM DDC for the indicated time. Phosphorylation of ERK1/2, p38 and Akt were determined by Western blotting analysis. The corresponding non-phosphorylated ERK1/2, p38, Akt and beta -actin were used for protein loading control. (B) Co-cultures were treated with or without 100 μM DDC for 24 h in the presence or absence of ERK1/2 inhibitor (U0126, 10 μM). MMP-1 protein levels were analysed by Western blotting. (C) Co-cultures were treated with or without 100 μM DDC for 24 h in the presence or absence of p38 inhibitor (SB203580, 10 μM). MMP-1 protein levels were analysed by Western blotting. (D) Co-cultures were treated with or without 100 μM DDC for 24 h in the presence or absence of Akt inhibitor (T3830, 50 μM). MMP-1 protein levels were analysed by Western blotting. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/23577625), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human AKT by Western Blot Enhancement of autophagy by SLAMF5 is not mediated by inhibition of the mTORC1 complex. (A) The autophagy-inducer rapamycin was applied to control or SLAMF5-silenced cells for 4 h. Autophagy was monitored by western blot analysis using an LC3-specific antibody. A representative blot of four independent experiments is shown on the left, and densitometry of LC3-II to beta -actin ratios is depicted on the right panel. (B) Cells were treated as described in panel (A), except that they were cultured in the presence of 20 nM bafilomycin A1 (BafA1) for the last 2 h. Data were analyzed as described in panel (A). (C)SLAMF5-silenced or control monocyte-derived dendritic cells were activated with LPS/IFN gamma, and phosphorylation of Akt and p70S6K was analyzed with immunoblotting. Representative blots of four independent experiments are shown. The phospho-proteins were normalized to the total levels of Akt1 and p70S6K. Data are expressed as mean ± SD (**p < 0.01 and ***p < 0.001; ns, not significant). Image collected and cropped by CiteAb from the following publication (https://journal.frontiersin.org/article/10.3389/fimmu.2018.00062/full), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human AKT by Western Blot p38 inhibitor SB203580 improves the up-regulation of MMP-1 by DDC through stimulating ERK1/2Co-cultures were treated with or without 100 μM DDC for 1 h in the presence or absence of p38 inhibitor (SB203580, 10 μM). Phosphorylation of ERK1/2, p38 and Akt in LX-2 cells of co-cultures were determined by Western blotting analysis. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/23577625), licensed under a CC-BY license. Not internally tested by R&D Systems.