Antibody data
- Antibody Data
- Antigen structure
- References [27]
- Comments [0]
- Validations
- Western blot [3]
- Immunocytochemistry [1]
- Immunohistochemistry [3]
- Flow cytometry [1]
- Other assay [10]
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Validation data
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- Product number
- 44-609G - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- AKT Pan Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Reactivity
- Human, Mouse
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Storage
- -20°C
Submitted references Pre-Exposure to Stress-Inducing Agents Increase the Anticancer Efficacy of Focused Ultrasound against Aggressive Prostate Cancer Cells.
Low-Dose Albendazole Inhibits Epithelial-Mesenchymal Transition of Melanoma Cells by Enhancing Phosphorylated GSK-3β/Tyr216 Accumulation.
Hyoscyamus albus nortropane alkaloids reduce hyperglycemia and hyperinsulinemia induced in HepG2 cells through the regulation of SIRT1/NF-kB/JNK pathway.
Cardiac Protective Effect of Kirenol against Doxorubicin-Induced Cardiac Hypertrophy in H9c2 Cells through Nrf2 Signaling via PI3K/AKT Pathways.
Effect and mechanism of downregulating the long-chain noncoding RNA TM4SF1-AS1 on the proliferation, apoptosis and invasion of gastric cancer cells.
Inhibitory Activity of Boesenbergia rotunda (L.) Mansf. Rhizome towards the Expression of Akt and NF-KappaB p65 in Acetic Acid-Induced Wistar Rats.
Wild‑type IDH1 affects cell migration by modulating the PI3K/AKT/mTOR pathway in primary glioblastoma cells.
Sevoflurane inhibits the proliferation and invasion of hepatocellular carcinoma cells through regulating the PTEN/Akt/GSK‑3β/β‑catenin signaling pathway by downregulating miR‑25‑3p.
Phosphorylation site S122 in estrogen receptor α has a tissue-dependent role in female mice.
Kaempferol Inhibits Zearalenone-Induced Oxidative Stress and Apoptosis via the PI3K/Akt-Mediated Nrf2 Signaling Pathway: In Vitro and In Vivo Studies.
A-kinase-interacting protein 1 promotes EMT and metastasis via PI3K/Akt/IKKβ pathway in cervical cancer.
Fat-Specific Protein 27 Regulation of Vascular Function in Human Obesity.
Preconception paternal alcohol exposure exerts sex-specific effects on offspring growth and long-term metabolic programming.
Programmed increases in LXRα induced by paternal alcohol use enhance offspring metabolic adaptation to high-fat diet induced obesity.
Thermogenesis, fatty acid synthesis with oxidation, and inflammation in the brown adipose tissue of ob/ob (-/-) mice.
Differential effects of angiotensin receptor blockers on pancreatic islet remodelling and glucose homeostasis in diet-induced obese mice.
Sixty years old is the breakpoint of human frontal cortex aging.
Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner.
A rich medium-chain triacylglycerol diet benefits adiposity but has adverse effects on the markers of hepatic lipogenesis and beta-oxidation.
Adverse effects of vitamin D deficiency on the Pi3k/Akt pathway and pancreatic islet morphology in diet-induced obese mice.
Pooled screening for antiproliferative inhibitors of protein-protein interactions.
A high-fish-oil diet prevents adiposity and modulates white adipose tissue inflammation pathways in mice.
p53 attenuates AKT signaling by modulating membrane phospholipid composition.
GPR30 activation is neither necessary nor sufficient for acute neuroprotection by 17β-estradiol after an ischemic injury in organotypic hippocampal slice cultures.
Enhanced pan-peroxisome proliferator-activated receptor gene and protein expression in adipose tissue of diet-induced obese mice treated with telmisartan.
Effect of whole-body vibration and insulin-like growth factor-I on muscle paralysis-induced bone degeneration after botulinum toxin injection in mice.
Signaling in sperm: toward a molecular understanding of the acquisition of sperm motility in the mouse epididymis.
Murad HY, Chandra PK, Kelly CA, Khurana N, Yu H, Bortz EP, Hong SN, Mondal D, Khismatullin DB
Antioxidants (Basel, Switzerland) 2022 Feb 9;11(2)
Antioxidants (Basel, Switzerland) 2022 Feb 9;11(2)
Low-Dose Albendazole Inhibits Epithelial-Mesenchymal Transition of Melanoma Cells by Enhancing Phosphorylated GSK-3β/Tyr216 Accumulation.
He Z, Lei S, Liang F, Tan L, Zhang W, Xie L, Zheng H, Lu Y
Journal of oncology 2021;2021:4475192
Journal of oncology 2021;2021:4475192
Hyoscyamus albus nortropane alkaloids reduce hyperglycemia and hyperinsulinemia induced in HepG2 cells through the regulation of SIRT1/NF-kB/JNK pathway.
Kowalczuk A, Bourebaba N, Kornicka-Garbowska K, Turlej E, Marycz K, Bourebaba L
Cell communication and signaling : CCS 2021 May 25;19(1):61
Cell communication and signaling : CCS 2021 May 25;19(1):61
Cardiac Protective Effect of Kirenol against Doxorubicin-Induced Cardiac Hypertrophy in H9c2 Cells through Nrf2 Signaling via PI3K/AKT Pathways.
Alzahrani AM, Rajendran P, Veeraraghavan VP, Hanieh H
International journal of molecular sciences 2021 Mar 23;22(6)
International journal of molecular sciences 2021 Mar 23;22(6)
Effect and mechanism of downregulating the long-chain noncoding RNA TM4SF1-AS1 on the proliferation, apoptosis and invasion of gastric cancer cells.
He C, Qi W, Wang Z
World journal of surgical oncology 2021 Jul 31;19(1):226
World journal of surgical oncology 2021 Jul 31;19(1):226
Inhibitory Activity of Boesenbergia rotunda (L.) Mansf. Rhizome towards the Expression of Akt and NF-KappaB p65 in Acetic Acid-Induced Wistar Rats.
Rosdianto AM, Puspitasari IM, Lesmana R, Levita J
Evidence-based complementary and alternative medicine : eCAM 2020;2020:6940313
Evidence-based complementary and alternative medicine : eCAM 2020;2020:6940313
Wild‑type IDH1 affects cell migration by modulating the PI3K/AKT/mTOR pathway in primary glioblastoma cells.
Shen X, Wu S, Zhang J, Li M, Xu F, Wang A, Lei Y, Zhu G
Molecular medicine reports 2020 Sep;22(3):1949-1957
Molecular medicine reports 2020 Sep;22(3):1949-1957
Sevoflurane inhibits the proliferation and invasion of hepatocellular carcinoma cells through regulating the PTEN/Akt/GSK‑3β/β‑catenin signaling pathway by downregulating miR‑25‑3p.
Cao Y, Lv W, Ding W, Li J
International journal of molecular medicine 2020 Jul;46(1):97-106
International journal of molecular medicine 2020 Jul;46(1):97-106
Phosphorylation site S122 in estrogen receptor α has a tissue-dependent role in female mice.
Ohlsson C, Gustafsson KL, Farman HH, Henning P, Lionikaite V, Movérare-Skrtic S, Sjögren K, Törnqvist AE, Andersson A, Islander U, Bernardi AI, Poutanen M, Chambon P, Lagerquist MK
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2020 Dec;34(12):15991-16002
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2020 Dec;34(12):15991-16002
Kaempferol Inhibits Zearalenone-Induced Oxidative Stress and Apoptosis via the PI3K/Akt-Mediated Nrf2 Signaling Pathway: In Vitro and In Vivo Studies.
Rajendran P, Ammar RB, Al-Saeedi FJ, Mohamed ME, ElNaggar MA, Al-Ramadan SY, Bekhet GM, Soliman AM
International journal of molecular sciences 2020 Dec 28;22(1)
International journal of molecular sciences 2020 Dec 28;22(1)
A-kinase-interacting protein 1 promotes EMT and metastasis via PI3K/Akt/IKKβ pathway in cervical cancer.
Zhang X, Liu S, Zhu Y
Cell biochemistry and function 2020 Aug;38(6):782-791
Cell biochemistry and function 2020 Aug;38(6):782-791
Fat-Specific Protein 27 Regulation of Vascular Function in Human Obesity.
Karki S, Farb MG, Sharma VM, Jash S, Zizza EJ, Hess DT, Carmine B, Carter CO, Pernar LI, Apovian CM, Puri V, Gokce N
Journal of the American Heart Association 2019 Jun 4;8(11):e011431
Journal of the American Heart Association 2019 Jun 4;8(11):e011431
Preconception paternal alcohol exposure exerts sex-specific effects on offspring growth and long-term metabolic programming.
Chang RC, Wang H, Bedi Y, Golding MC
Epigenetics & chromatin 2019 Jan 22;12(1):9
Epigenetics & chromatin 2019 Jan 22;12(1):9
Programmed increases in LXRα induced by paternal alcohol use enhance offspring metabolic adaptation to high-fat diet induced obesity.
Chang RC, Thomas KN, Bedi YS, Golding MC
Molecular metabolism 2019 Dec;30:161-172
Molecular metabolism 2019 Dec;30:161-172
Thermogenesis, fatty acid synthesis with oxidation, and inflammation in the brown adipose tissue of ob/ob (-/-) mice.
Martins FF, Bargut TCL, Aguila MB, Mandarim-de-Lacerda CA
Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft 2017 Mar;210:44-51
Annals of anatomy = Anatomischer Anzeiger : official organ of the Anatomische Gesellschaft 2017 Mar;210:44-51
Differential effects of angiotensin receptor blockers on pancreatic islet remodelling and glucose homeostasis in diet-induced obese mice.
Graus-Nunes F, Marinho TS, Barbosa-da-Silva S, Aguila MB, Mandarim-de-Lacerda CA, Souza-Mello V
Molecular and cellular endocrinology 2017 Jan 5;439:54-64
Molecular and cellular endocrinology 2017 Jan 5;439:54-64
Sixty years old is the breakpoint of human frontal cortex aging.
Cabré R, Naudí A, Dominguez-Gonzalez M, Ayala V, Jové M, Mota-Martorell N, Piñol-Ripoll G, Gil-Villar MP, Rué M, Portero-Otín M, Ferrer I, Pamplona R
Free radical biology & medicine 2017 Feb;103:14-22
Free radical biology & medicine 2017 Feb;103:14-22
Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner.
Tam KJ, Dalal K, Hsing M, Cheng CW, Khosravi S, Yenki P, Tse C, Peacock JW, Sharma A, Chiang YT, Wang Y, Cherkasov A, Rennie PS, Gleave ME, Ong CJ
Oncotarget 2017 Feb 7;8(6):9617-9633
Oncotarget 2017 Feb 7;8(6):9617-9633
A rich medium-chain triacylglycerol diet benefits adiposity but has adverse effects on the markers of hepatic lipogenesis and beta-oxidation.
Chamma CM, Bargut TC, Mandarim-de-Lacerda CA, Aguila MB
Food & function 2017 Feb 22;8(2):778-787
Food & function 2017 Feb 22;8(2):778-787
Adverse effects of vitamin D deficiency on the Pi3k/Akt pathway and pancreatic islet morphology in diet-induced obese mice.
Borges CC, Salles AF, Bringhenti I, Souza-Mello V, Mandarim-de-Lacerda CA, Aguila MB
Molecular nutrition & food research 2016 Feb;60(2):346-57
Molecular nutrition & food research 2016 Feb;60(2):346-57
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
Nature chemical biology 2016 Apr;12(4):275-81
A high-fish-oil diet prevents adiposity and modulates white adipose tissue inflammation pathways in mice.
Bargut TC, Mandarim-de-Lacerda CA, Aguila MB
The Journal of nutritional biochemistry 2015 Sep;26(9):960-9
The Journal of nutritional biochemistry 2015 Sep;26(9):960-9
p53 attenuates AKT signaling by modulating membrane phospholipid composition.
Rueda-Rincon N, Bloch K, Derua R, Vyas R, Harms A, Hankemeier T, Khan NA, Dehairs J, Bagadi M, Binda MM, Waelkens E, Marine JC, Swinnen JV
Oncotarget 2015 Aug 28;6(25):21240-54
Oncotarget 2015 Aug 28;6(25):21240-54
GPR30 activation is neither necessary nor sufficient for acute neuroprotection by 17β-estradiol after an ischemic injury in organotypic hippocampal slice cultures.
Lamprecht MR, Morrison B 3rd
Brain research 2014 May 14;1563:131-7
Brain research 2014 May 14;1563:131-7
Enhanced pan-peroxisome proliferator-activated receptor gene and protein expression in adipose tissue of diet-induced obese mice treated with telmisartan.
Penna-de-Carvalho A, Graus-Nunes F, Rabelo-Andrade J, Mandarim-de-Lacerda CA, Souza-Mello V
Experimental physiology 2014 Dec 1;99(12):1663-78
Experimental physiology 2014 Dec 1;99(12):1663-78
Effect of whole-body vibration and insulin-like growth factor-I on muscle paralysis-induced bone degeneration after botulinum toxin injection in mice.
Niehoff A, Lechner P, Ratiu O, Reuter S, Hamann N, Brüggemann GP, Schönau E, Bloch W, Beccard R
Calcified tissue international 2014 Apr;94(4):373-83
Calcified tissue international 2014 Apr;94(4):373-83
Signaling in sperm: toward a molecular understanding of the acquisition of sperm motility in the mouse epididymis.
Vadnais ML, Aghajanian HK, Lin A, Gerton GL
Biology of reproduction 2013 Nov;89(5):127
Biology of reproduction 2013 Nov;89(5):127
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Supportive validation
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- Extracts of 3T3L1 cells were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer for one hour at room temperature, then incubated with a 1:1,000 (1) or 1:500 (2) dilution of the AKT (Pan) Polyclonal Antibody, Rabbit for two hours at room temperature in a 3% BSA-TBST buffer. After washing, the membrane was incubated with goat F (ab’) 2 anti-rabbit IgG HRP-conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ method.
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- Western blot analysis of AKT-1 was performed by loading 30 µg of U-87 MG (Lane 1), U-87 MG - AKT-1 knockout (Lane 2) whole cell lysate. The blot was probed with Anti-AKT Pan Antibody (Product # 44-609G, 1 µg/mL) Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to AKT-1.
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- Western blot analysis of AKT pan was performed by loading 20 µg of HeLa (lane1), MCF7 (lane2), NIH\u0003T3 (lane3), THP-1 (lane4) and U-87 MG (lane5) cell lysate using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. AKT pan was detected at 55 kDa using AKT pan Rabbit Polyclonal Antibody (Product # 44-609G) at 1:500 dilution in 5% skim milk at 4°C overnight on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).
Supportive validation
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- Immunofluorescence analysis of AKT pan was done on 70% confluent log phase U2OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with AKT pan Rabbit Polyclonal Antibody (Product # 44-609G) at 1:250 dilution in 0.1%BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.
Supportive validation
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- Immunohistochemistry analysis of AKT (Pan) 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 10mM 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 a AKT (Pan) Rabbit Polyclonal Antibody (Product # 44-609G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an 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.
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- Immunohistochemistry analysis of AKT (Pan) 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 10mM 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 a AKT (Pan) Rabbit Polyclonal Antibody (Product # 44-609G) 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 an 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.
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- Immunohistochemistry analysis of AKT (Pan) showing staining in the cytoplasm and nucleus of paraffin-embedded human breast carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM 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 a AKT (Pan) Rabbit Polyclonal Antibody (Product # 44-609G) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an 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
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- Flow cytometry analysis of AKT pan was done on HEK-293 cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with AKT pan Rabbit Polyclonal Antibody (44609G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 µg/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.
Supportive validation
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- Figure 6 p53-mediated SCD repression attenuates AKT activation A. HCT116 p53 +/+ and p53 -/- cells were treated with 5 muM nutlin-3 for 72 h and pAKT (S473) and total AKT levels were assessed by western blotting. GAPDH was used as loading control. B. AKT and pAKT (S473) levels in tissues from hypo Mdm2 and control mice ( n = 5). The ratio pAKT/AKT is expressed relative to control, p = 0, 07, unpaired t test. C. Re-introduction of SREBP1 restores pAKT in nutlin-3-treated cells. HCT116 p53 +/+ cells were infected with an adenovirus encoding SREBP1c or empty virus. Four h after infection, cells were treated 5 muM nutlin-3 for 72 h. pAKT and total AKT levels were assessed by western blotting. The blot was cropped to show the conditions of interest. D and F. Oleic acid (OA) rescues the effect of p53 on pAKT by restoring phospholipid profiles. HCT116 cells were treated with OA (100 muM) in combination with 5 muM nutlin-3 (Nut). Phospholipid profiles are presented as described in the legend to Figure 1 . Representative blots of total AKT and pAKT (S473) are shown. The graph presents the average pAKT/AKT ratio of two independent samples. * p < 0.05, ** p < 0.01 by ANOVA. E and G. PI36:2 restores pAKT (S473) levels in nutlin-3-treated cells. HCT116 cells were treated with 5 muM nutlin-3 alone (Nut) or in combination with PI36:2 at 10 muM. After 72 h, cells pellets were collected for lipid or western blot analysis. Phospholipid profiles were presented as described in the legend to Figu
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- Figure 4 KFL triggers the PI3K/Akt pathway in ZEA-induced HepG2 cells. ( A ) Cells were treated with KFL (10, 25 and 50 uM) followed by ZEA (40 uM) for 24 h. After the treatment, whole-cell lysates were exposed to Western blotting with anti-pPI3K and anti-pAkt antibodies. Total PI3K and Akt levels were measured as loading controls. ( B ) Cells were pre-treated with a PI3K/Akt inhibitor (LY294002, 30 muM) for 2 h, followed by KFL (50 muM) and/or ZEA (40 muM) for 24 h. Western blot was performed to detect the pAkt, HO-1 and Nrf2 levels by anti-pAkt, anti-HO-1 and anti-Nrf2 abs. Data are represented as the mean +- SD of triplicate values ( n = 3), and * p < 0.05 represents noteworthy discrepancies compared with the control. # p < 0.05 represents significant variations compared with the ZEA alone and KFL with ZEA treatment groups.
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- Figure 3 KRL enhances cell survival mechanism. H9c2 cells were cultured in serum-free media for 3 h, followed by treatment with KRL for 2 h before or after DOX treatment, respectively. ( A , B ) KRL activates the IGF1R-mediated survival pathway in H9c2 cells. The expression of p-IGF1R, PI3K, p-PI3K, AKT, and p-AKT was analyzed by Western blotting. beta-actin was used as the internal control. ( C ) Representative Western blots showing the changes in MAPK signaling proteins (pP38 and pJNK) in H9c2 cells. Data are represented as the mean +- SD of triplicate values ( n = 3) and * p < 0.05 represents significant variations compared with the control. # p < 0.05 represents significant variations as compared to DOX alone and KRL with DOX treatment groups.
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- Representative Western blot analysis of white adipose tissue for IRS-1 (A), p-Akt (B), TC10 (C) and GLUT4 (D) The average values were determined, and equal protein loading was confirmed by probing the blots with beta-actin antibodies. Data are reported as the means +- SEM, n = 5 in each group. Significant differences between the groups are indicated by symbols ( P < 0.05) and were determined by a one-way ANOVA and the Holm-Sidak post hoc test.
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- Fig. 7 Effect of total calystegines on collapsed insulin signalling pathway in HI/HG HepG2 cells. a Quantitative analysis of insulin signalling-related proteins expression using western blot. b Representative blots images of insulin signalling-related proteins profiling. c Relative genes expression of key insulin signalling regulators. Representative data from three independent experiments are shown +- SD (n = 3). An asterisk (*) indicates a comparison of IR group to untreated healthy cells. A hashtag (#) indicates a comparison of IR group pre-treated with calystegines to IR untreated healthy cells. */# p < 0.05, **/## p < 0.01, ***/### p < 0.001. HepG2_HE: HepG2 healthy untreated cells; HepG2_IR: Insulin resistant HepG2 cells exposed to high concentrations of insulin and glucose. HepG2_IR- H. albus _Caly: Insulin resistant HepG2 cells exposed to high concentrations of insulin and glucose and pre-treated with 250 mug/ml calystegines extracted from Hyoscyamus albus seeds
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- Figure 4 ABZ treatment downregulates the snail expression in melanoma cells by increasing the accumulation of phosphorylated GSK-3 beta /Tyr216. (a) The relative transcription levels of Snail in the ABZ-treated (0.4 mu M) and control groups of A375 (left) and B16-F10 (right) melanoma cells were measured by RT-qPCR, with beta -actin as the internal control. (b) The expression of transcription factor Snail in A375 (left) and B16-F10 (right) cells was detected by western blot analysis, with beta -actin as the internal reference protein. (c-d) The expression levels of cytoplasmic proteins AKT, pAKT, GSK-3 beta , pGSK-3 beta (Ser9/Tyr216) and Snail, and nuclear protein pSnail in A375 and B16-F10 cells were also determined by western blotting, with beta -actin and PCNA as the internal controls for the cytoplasmic and nuclear proteins, respectively. The histograms show the relative density of AKT/pAKT, GSK-3 beta /pGSK-3 beta (Ser9/Tyr216), and Snail/p-Snail. (e) A375 cells were cotreated with or without MG132 and 0.4 mu M ABZ for 24 h western blot (up) was used to detect the expression levels of AKT, pGSK-3 beta /Tyr216, Snail, N-cadherin, and E-cadherin in the cytoplasm of A375 cells. The histogram (bottom) shows the relative density of AKT, pGSK-3 beta /Tyr216, Snail, E-cadherin, and N-cadherin. (f) Histogram showing the relative expression intensity of pGSK-3 beta (Ser9/Tyr216) and pAKT after immunohistochemical staining of mouse metastatic lung cancer tissues. Scale bars = 100
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- Figure 6 Effect of FUS, alone and in combination with CDDO-me and/or nelfinavir, on both total and activated NF-kappaB and Akt protein levels in DU145 cells. The effect of combined treatment with nelfinavir and CDDO-me (CD), alone or in combination with FUS (N + CD + H4), on both total and activated (phosphorylated) NF-kappaB p65 ( A - C ) and Akt ( D - F ) protein levels is shown. Representative immunoblot images are in the top panel, along with the loading control (GAPDH). Bar graphs in each of the bottom panels of A-F show normalized densitometric values. Data are representative of three independent experiments ( n = 3). Significant changes from untreated controls or individual treatment groups are shown as p -values (* p < 0.05; ** p < 0.01; and *** p < 0.001). Unlike the recurrence of NF-kappaB and Akt levels in DU145 cells exposed to FUS alone (cf. Supplemental Figure S1 ), our three-treatment combination (N + CD + H4) abrogated the reactivation of these two crucial transcription factors.
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- Figure 3 Insulin-mediated activation of eNOS (endothelial NO synthase) and AKT (protein Kinase B) in response to recombinant FSP 27 in visceral depot. A , Representative visceral adipose tissue immunoblot demonstrating severe impairment in insulin-mediated activation of eNOS and AKT in visceral fat. After 24 hours of rFSP 27 (recombinant FSP 27) exposure, insulin-mediated activation is restored. B , Quantification of percent change in insulin-mediated activation of eNOS at baseline and after 24 hours of treatment with rFSP 27 in the visceral depot. C , Quantification of percent change in insulin-mediated activation of AKT at baseline and after 24 hours of treatment with rFSP 27 in the visceral depot (n=10, P
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- Figure 5 Insulin-mediated activation of eNOS (endothelial NO synthase) and AKT (protein Kinase B) in response to si RNA -mediated knockdown of FSP 27 in the subcutaneous depot. A , Representative immunoblot demonstrating insulin-mediated activation of eNOS and AKT in subcutaneous fat under scrambled si RNA (small interfering RNA ) conditions and after knockdown of FSP 27 by si RNA . B , Quantification of percent change in insulin-mediated activation of eNOS at baseline and after si RNA -mediated knockdown of FSP 27 in the subcutaneous fat depot. C , Quantification of percent change in insulin-mediated activation of AKT at baseline and after si RNA -mediated knockdown of FSP 27 in subcutaneous fat depot (n=10, P
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- 6 FIGURE AKIP1 Suppresses PTEN during the EMT. A, HeLa and CaSki cervical cancer cells were transfected with a control siRNA (siNC) or AKIP1 siRNA. The cell lysates were collected and the protein expression of PTEN, AKIP1, p-Akt, total Akt, and beta-Actin was detected using western blot. B, Stable AKIP1-expressing cells were transfected with EV, wild-type PTEN, or G129E-mutant PTEN plasmids. At 48 hours after transfection, cell lysates were harvested after cell fractionation. Levels of indicated proteins were determined using western blot analysis