44-621G

antibody from Invitrogen Antibodies

Targeting: AKT1 AKT, PKB, PRKBA, RAC, RAC-alpha

Provider product page for 44-621G

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [42]
Comments [0]
Validations
Immunocytochemistry [2]
Other assay [26]

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Product number: 44-621G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-AKT1 (Ser473) Monoclonal Antibody (14-6)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: This product contains enough material for 10 mini-blots. The immunogenic sequence is conserved among multiple species including human, mouse, and rat in all three isoforms: Akt1, Akt2, and Akt3. Others species (100% homologous) have not been tested. Recommended positive controls include NIH-3T3 cells stimulated with PDGF and 3T3-L1 adipocytes stimulated with OSM and IFN-gamma.
Reactivity: Human, Mouse
Host: Rabbit
Isotype: IgG
Antibody clone number: 14-6
Vial size: 100 µL
Concentration: 0.5 mg/mL
Storage: -20°C

AKT1 protein structure - 44-621G shown in red.

Supportive validation

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Supportive validation

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Supportive validation

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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

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Experimental details: 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

Supportive validation

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Experimental details: Figure 4 PPM1A overexpression affects cell cycle and survival markers. ( A ) PC6-3 cells overexpressing wt or mutant PPM1A were incubated with or without Tet for the indicated times. The cells were then harvested and lysed. Cell extracts were analyzed by western blot for levels of phosphorylated AKT, total AKT and PPM1A. Note that PPM1A mutant migrates faster than the wt on the gel ( [10] ) and that even in the absence of Tet there was a slight expression due to leakiness of PPM1A mutant. PPM1A wt cells were analyzed in the same manner for levels of ( B ) cleaved caspase 3 and p38. ( C ) PC6-3 cells overexpressing PPM1A were incubated with or without Tet for 24 hours. The cells were then harvested and lysed and cell extracts were analyzed for levels of p53, Mdm2, PPM1A and p38 by western blot. The numbers represent the ratio of p53/p38 (0.1, 0.07) and Mdm2/p38 levels (0.5, 0.5). The blots presented in ( A ) ( B ) and ( C ) are from representative experiments out of three performed.

Supportive validation

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Experimental details: Figure 8 PPM1A over and underexpression and cell signaling during PC6-3 cells differentiation. ( A ) PPM1A wt cells were incubated with or without Tet for 24 hours and then NGF was added for the indicated times. ( B ) PC6-3 PPM1A knock-down cells and control cells were incubated with NGF for the indicated times. The cells were harvested and lysed. Cell extracts were analyzed by western blot using the indicated antibodies. Please note that the AKT and PPM1A panels in ( B ) are from different gels of the same experiments. The graphs represent quantification of the blots shown in ( A ) and ( B ) . The results shown are from representative experiments out of 3 independent that were performed.

Supportive validation

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Experimental details: Fig 3 (A) The effect of DMSO on modulating the activation of cell signaling pathways in response to E . coli- stimulation of human blood monocytes. (B ) The effect of cell signaling inhibitors on IL-6 production form E . coli -challenged human monocytes. Inhibitor concentrations were chosen based on the lowest levels that markedly inhibited their pathways. *denotes significant inhibition (P

Supportive validation

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Experimental details: Figure 12 ( A ) Western blot representing inhibitory effect of treatments on expression of p-AKT protein in A549 cells. ( B) PFD-D-Lip significantly suppresses p-AKT expression in A549 cells. Cell lysates were prepared following 24-hour treatment of A549 cells with control, PFD and PFD-D-Lip (0.125 mg/mL) and were subjected to western blot analyses using antibodies against p-AKT and actin as a loading control, as indicated. ( C ) Western blot representing inhibitory effect of treatments on expression of beta-catenin protein in A549 cells. ( D ) PFD-D-Lip suppresses beta-catenin expression in A549 cells. Cell lysates were prepared following 72-hour treatment of A549 cells with control, PFD and PFD-D-Lip (0.25 and 0.5 mg/mL) and were subjected to western blot analyses using antibodies against beta-catenin and actin as a loading control, as indicated. The western blot densities were determined and plotted as relative band intensity against treatments. Western blot bands represent representative image of n = 3. Data represent mean +- SD ( n = 3). * p (alpha < 0.05), ** p (alpha < 0.01).

Supportive validation

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Experimental details: 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.

Supportive validation

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Experimental details: Figure 5 Effects of GH and IGF-1 treatments upon activation of the PI3K/Akt and MAPK/ERK1/2 signaling pathways and Bcl-2 expression after a hypoxic-ischemic injury in primary cerebellar cultures. Cells were maintained under either normoxic conditions (Nx), submitted to hypoxic-ischemic injury for 12 h (HI) and then reoxygenated for 24 h (HI + Ox), or treated with 1 nM GH or 40 nM IGF-1 after (denoted by A) 12h of HI, along the 24 h of reoxygenation period (HI + Ox GH-A; HI + Ox IGF-1-A). The upper panels show Western blots with specific immunoreactive bands corresponding to the following proteins: ( A ) total Akt (60 kDa) or pAkt (S473, 60 kDa); ( C ), ERK1/2 (44 kDa) or pERK1/2 (T202/Y204, 44 kDa); ( E ), Bcl-2 (27 kDa); and GAPDH (panels ( A , C , E ), 37 kDa) as loading control. ( B , D , F ) show densitometric (% O.D.) analysis of changes observed in ( A , C , E ). Bars represent the mean +- SEM, n = 5 independent experiments performed in duplicate. Groups with different letters are significantly different by one-way ANOVA and Tukey's post hoc test ( p < 0.01).

Supportive validation

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Experimental details: 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.

Supportive validation

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Experimental details: Figure 6 D-Chiro-Ins does not modulate the expression of Akt. Mature SGBS adipocytes cells were treated with D-Chiro-Ins 10 -8 M, E2 10 -9 M or INS 10 -7 M, and combinations for 24 h. Cell lysates were immunoblotted with an anti-Akt antibody, anti-p-Akt antibody, and anti-GAPDH antibody. (A) Panels show representative blot of Akt, p-Akt Ser473 and GAPDH. (B-D) Analysis of the relative expression level of Akt, p-Akt and the ratio p-Akt/Akt. The expression level of Akt and p-Akt was normalized with the protein amount of GAPDH, relative to the control. Values are presented as mean +- SEM of three independent experiments. Results were analyzed statistically by one-way RM ANOVA followed by Dunnett''s multiple comparisons test (*p < 0.05, **p < 0.01 versus control). D-Chiro-Ins, D-chiro-Inositol; E2, 17beta-estradiol, INS, insulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Supportive validation

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Experimental details: Fig. 4 Phlpp1-deletion (A) promoted Akt phosphorylation, (B) increased cell proliferation, and (C) decreased apoptosis 3 days after needle puncture injury. a ) Immunohistochemistry staining for pAkt demonstrated elevated Akt phosphorylation in the AF of Phlpp1 KO ( n = 5) compared to WT mice ( n = 7) at baseline. Injury further caused an increase of pAkt in Phlpp1 KO mice, but not in WT mice 3 days after needle puncture. ROI (region of interest) is the magnification of the region in the black boxes. Arrows indicate positive cells. Positive and negative controls were used for antibody validation. b ) EdU and c ) TUNEL assay of IVD sections 3 days after needle puncture. EdU-positive cells were increased in injured AF of Phlpp1 KO mice, compared to the other three groups ( n = 7 per group). TUNEL-positive cells were increased significantly in AF tissues of WT mice, but not in Phlpp1 KO mice 3 days after injury ( n = 4 per group). White dotted lines denote the border between the NP and AF compartments. Yellow boxes mark the magnified ROI. Arrows denote positive cells. Nuclei were stained with DAPI. Two-way ANOVAs were used to assess the effect of injury and genotype

Supportive validation

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Experimental details: Figure 4. CDCA4 affected cell cycle and AKT/mTOR signaling pathway. (a-b) GSEA was utilized to identify the relative signaling pathway associated with high CDCA4 expression. (c-f) The effects of CDCA4-knockdown (c-d) and -overexpression (e-f) on the AKT/mTOR relative markers and Cyclin D1 protein were evaluated using western blot analysis. (d) Quantification of c. (f) Quantification of e. ** p < 0.01 vs . si-NC or vector group.

Supportive validation

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Experimental details: Figure 2 Kinetics of Erk1/2 and Akt phosphorylation in the presence of idInlBs. A total of 100 ng/mL idInlBs were added to HEp-2 cells. Cells were lysed at pointed time points and probed with anti-phospho-Erk1/2 and anti-phospho-Akt antibodies. The curves show digitized data from three independent experiments. * p < 0.05.