- AHO1112 - Invitrogen Antibodies AKT1 antibody

Submitted references Cancer-associated fibroblast-derived exosomal microRNA-20a suppresses the PTEN/PI3K-AKT pathway to promote the progression and chemoresistance of non-small cell lung cancer.

Shi L, Zhu W, Huang Y, Zhuo L, Wang S, Chen S, Zhang B, Ke B
Clinical and translational medicine 2022 Jul;12(7):e989

In Vitro Wound Healing Potential of Photobiomodulation Is Possibly Mediated by Its Stimulatory Effect on AKT Expression in Adipose-Derived Stem Cells.

Rajendran NK, Houreld NN, Abrahamse H
Oxidative medicine and cellular longevity 2021;2021:6664627

Loss of FBXO31-mediated degradation of DUSP6 dysregulates ERK and PI3K-AKT signaling and promotes prostate tumorigenesis.

Duan S, Moro L, Qu R, Simoneschi D, Cho H, Jiang S, Zhao H, Chang Q, de Stanchina E, Arbini AA, Pagano M
Cell reports 2021 Oct 19;37(3):109870

Regulation of H-Ras-driven MAPK signaling, transformation and tumorigenesis, but not PI3K signaling and tumor progression, by plasma membrane microdomains.

Michael JV, Wurtzel JG, Goldfinger LE
Oncogenesis 2016 May 30;5(5):e228

Pooled screening for antiproliferative inhibitors of protein-protein interactions.

Nim S, Jeon J, Corbi-Verge C, Seo MH, Ivarsson Y, Moffat J, Tarasova N, Kim PM
Nature chemical biology 2016 Apr;12(4):275-81

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of AKT was performed by loading 20 µg of Raji (lane1), NIH/3T3 (lane2), HeLa (lane3), MCF7 (lane4), Jurkat (lane5) and A549 (lane6) cell lysates using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. AKT was detected at ~60 kDa using AKT Mouse Monoclonal Antibody (Product # AHO1112) at 1-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of AKT showing staining in the cytoplasm and nucleus of paraffin-embedded human lung squamous carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with AKT monoclonal antibody (Product # AHO1112) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of AKT showing staining in the cytoplasm and nucleus of paraffin-embedded human prostate carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with AKT monoclonal antibody (Product # AHO1112) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of AKT showing staining in the cytoplasm and nucleus of paraffin-embedded mouse prostate tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with AKT monoclonal antibody (Product # AHO1112) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using a HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Effect of photobiomodulation on AKT in normal, wounded, diabetic, and diabetic wounded ADSCs irradiated at a wavelength of 660 and 830 nm with a fluence of 5 J/cm 2 . AKT is stained green with FITC, and nuclei are stained blue with DAPI. Both the irradiated and nonirradiated groups displayed positive staining for AKT proteins. Irradiated cells expressed more AKT protein compared to the nonirradiated groups. Red arrows indicate the presence of AKT. Magnification = x100.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3. Depletion of FBXO31 regulates ERK and AKT activity and promotes cell growth in prostate cells (A) Parental and FBXO31 KO RWPE1 cells were treated with vehicle or the DUSP6 inhibitor BCI for the indicated days, and cell viability was determined by ATP-Glo assay. Graph was generated using Prism 8 (mean +- SD; n = 8). (B) VCAP cells stably transduced with a retroviral system expressing doxycycline-inducible FBXO31 or a control vector were treated with doxycycline for the indicated times. WCLs were immunoblotted as indicated. (C) RWPE1 cells were treated with various concentrations of BCI for 6 h and then analyzed by immunoblotting for the indicated proteins. (D) FBXO31 wild-type or KO RWPE1 cells were cultured in medium without EGF for the indicated times (withdrawal [W/D]) and then harvested and analyzed by immunoblotting. (E) FBXO31 wild-type or KO RWPE1 cells were cultured in regular medium or in medium without EGF (W/D). Where indicated, cells were treated with the DUSP6 inhibitor BCI. GTP-bound RAS was quantified using RAF1 Ras-binding domain (RBD) pull-down assay. Western blotting images are representative of 3 independent experiments, and the intensity of bands was quantified by ImageJ. The columns represent the relative levels of RAS-GTP as normalized to the levels of total RAS protein (mean +- SD; n = 3 independent experiments) (F) FBXO31 wild-type or KO RWPE1 cells were cultured in regular medium or medium without EGF. Relative cell viability was measured by

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 4 FIGURE miR-20a promotes in vitro cisplatin resistance and proliferation in non-small-cell lung cancer (NSCLC) cells. (A) Levels of miR-20a expression were assessed in HCC827 and H1299 cells following miR-20a mimic transfection, miR-NC mock transfection or control treatment. The viability (B and C) and DDP IC50 values (D) for NSCLC cell lines treated as in (A) were measured. The proliferation (E and F), colony formation (G and H) and Hoechst staining (I and J) results for NSCLC cell lines treated as in (A). (K and L) Western blotting was used to assess PI3K-p85, PI3K-p100, PI3K, pAKT and AKT expression in the indicated cell lines in the presence or absence of DDP treatment, with GAPDH as a normalization control. Outcomes are means +- standard deviation (SD) (* p < .05, ** p < .01, *** p < .001 vs. control; ## p < .01, ### p < .001 vs. mock + DDP)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 8 FIGURE miR-20a-containing exosomes derived from cancer-associated fibroblasts (CAFs) regulate the PI3K/AKT pathway in vivo to promote non-small-cell lung cancer (NSCLC) tumour growth and chemoresistance. Nude mice were subcutaneously implanted with HCC827 cells treated using PBS, normal tissue-associated fibroblasts (NAFs), CAF or CAFs-shRab27a. Mice were then randomized into DDP treatment and untreated control groups. (A) Images of representative xenograft tumours. (B) Tumour volumes were analysed once weekly beginning 1 week after implantation. (C) The expression of miR-20a was assessed via qPCR in tumours from untreated mice. (D) The expression of miR-20a was assessed via qPCR in tumours from DDP-treated mice. (E) PTEN, PI3K-p85, PI3K-p100, PI3K, pAKT and AKT were analysed via Western blotting in tumour-bearing mice. (F) Tumours from xenograft model mice were subjected to Ki-67 and TUNEL staining to analyse apoptotic cell death (200x), with the positive staining area being quantified in (G and H). Results are representative data from triplicate experiments, and outcomes are means +- standard deviation (SD) (* p < .05; ** p < .01; *** p < .001)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: 9 FIGURE Exosomes derived from cancer-associated fibroblasts (CAFs) induce tumour growth and chemoresistance, and activate PI3K/AKT pathway in a nude mouse non-small-cell lung cancer (NSCLC) model. HCC827 cells were processed with exosomes derived from normal tissue-associated fibroblasts (NAFs) or CAFs were subcutaneously injected into nude mice, which were treated or untreated with DDP. (A) Subcutaneous tumours in each group were excised and displayed. (B) Tumour volume was measured at the end of 3, 7, 14, 21 and 28 days. (C) qPCR analysis of miR-20a expression in tumours. (D) Western blotting analysis of PTEN, PI3K-p85, PI3K-p100, PI3K, pAKT and AKT. Outcomes are means +- standard deviation (SD) (* p < .05; ** p < .01; *** p < .001)

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