BioPath Online

Pathway Focus: The AKT Pathway

Multiplex AKT assay—superior performance, high quality, fast and easy protocols
Multiplex AKT Assay—Superior performance, high quality, fast and easy protocols
PDGF-mediated activation of Akt signalingPDGF-Mediated Activation of AKT Signaling—Open the door to cell survival, protein synthesis, cell cycle, angiogenesis
Akt protein measurement using phosphoELISA™AKT Protein Measurement Using phosphoELISA™—Quickly detect and quantify phosphorylation site-specific proteins
ABfinity™ antibodies and the AKT pathway—the next generation of antibodiesABfinity™ Antibodies and the AKT Pathway—The next generation of antibodies
Live-cell imaging of cell cycle and divisionLive-Cell Imaging of Cell Cycle and Division—For the first time, real time
New Antibodies

New Immunoassays

New Molecular Probes® Products

Multiplex AKT Assay—Superior Performance, High Quality, Fast and Easy Protocols

  • Superior performance—accurate, reproducible, and sensitive quantitation of Akt pathway proteins
  • High quality—in-house manufactured antibodies ensure specificity and sensitivity
  • Fast and easy protocols—perform and analyze data in less than one day

Currently, Akt and its pathway are one of the most actively studied kinases and kinase pathways in basic research and drug development. Activated Akt is a key signaling mediator for cell growth, cell survival (anti-apoptosis), cell-cycle progression, differentiation, transcription, translation, and glucose metabolism.

Get accurate quantitation of 7 AKT pathway proteins with the AKT Total and Phospho 7-Plex Panel, exclusively from Invitrogen. This panel can detect protein levels, needs only a small sample, and can be run in ~4 hr. Validation includes bead and protein specificity, precision, intra- and inter-assay of <10%, parallelism between recombinant and natural samples, linearity of dilution, recovery (>80%), and comparison to ELISA (>90%). Premixed multiplex kits include General Buffer Reagent Kit and Antibody Bead Kit components, with reagents premixed for the number of markers.
MCF7 cells were treated with IGF-1

Figure 1. MCF7 cells were treated with IGF-1 (Panel I), and IR-transfected CHO-T cells were treated with insulin (Panel II). Cells were serum-starved for 15 hr in the presence of sodium vanadate and either left untreated or treated with 100 ng/mL of IGF-1 for 1 hr (Panel I) or with 100 nM of insulin for 15 min (Panel II). The levels of phospho (graphs A & D) and total (graphs B & E) proteins of the 7 analytes were determined. Data presented in graphs C & F show results of normalizing the levels of each phosphoprotein to its corresponding total protein.


ProductSpecies
Markers
 Cat. No.
 
Akt Pathway Phospho 7-Plex PanelHuman, mouse, rat
Akt [pS473], GSK-3β [pS9], IRS-1 [pS312], IGF-1R [pYpY1135/1136], IR [pYpY1162/1163], p70S6K [pTpS421/424], PRAS40 [pT246]
LHO0001
Akt Pathway Total 7-Plex PanelHuman, mouse, rat
Akt, GSK-3β, IGF-1R, IR, IRS-1, p70S6K, PRAS40
LHO0002

PDGF-Mediated Activation of AKT Signaling

  • Open the door to cell survival, protein synthesis, cell cycle, angiogenesis, etc.
  • Facilitate Akt pathway activation—recombinant PDGF-AA, PDGF-BB, and PDGF-AB
  • Enhanced signal studies—variety of other growth factors

Platelet-derived growth factor (PDGF) contributes to cell survival, movement, proliferation, angiogenesis, etc. Two classical growth factors in the PDGF family are PDGF-A and PDGF-B, active as homodimers and as the heterodimer PDGF-AB. Active growth factors bind to two receptors, PDGFR-α and PDGFR-β.
Once bound, the receptors lead to activation of various pathways associated with receptor tyrosine kinase signaling, including the Akt pathway. Upon receptor activation, PI3K interacts with the receptor and activates Akt, promoting downstream events.

Invitrogen offers recombinant PDGF-AA, PDGF-BB, and PDGF-AB to facilitate Akt pathway activation. In addition, we offer other growth factors to study Akt signaling.

Learn more at www.invitrogen.com/proteins

Immunofluorescence staining

Figure 1. Immunofluorescence staining.
Serum-starved NIH3T3 cells left untreated (left) or treated with PDGF (right) were fixed prior to immunostaining with the Akt [pS473] rabbit monoclonal antibody.The signal was detected with an anti-rabbit FITC-conjugated secondary antibody. The data show that the antibody detected phosphorylated Akt in PDGF-treated NIH3T3 cells. Cells were counterstained with DAPI.


Description
Species
Qty.
Cat. No.
 
PDGF-AA
Hu
10 µg
PHG0035
PDGF-AB
Hu
10 µg
PHG0134
PDGF-BB
Hu
5 µg
PHG0044
PDGF-BB
Hu
10 µg
PHG0045
PDGF-BB
Hu
50 µg
PHG0046
PDGF-BB
Hu
100 µg
PHG0041
PDGF-BB
Hu
1 mg
PHG0043
PDGF-BB
Ms
10 µg
PMG0044
PDGF-BB
Ms
25 µg
PMG0045
PDGF-BB
Ms
100 µg
PMG0041
PDGF-BB
Ms
1 mg
PMG0043

AKT Protein Measurement Using phosphoELISA™ Kits

  • Sandwich ELISA kits quickly detect and quantify site-specific phosphoproteins in cell lysates
  • Total kits normalize protein content of the sample
  • High throughput of samples in a 96-well format

AKT can be activated by a diverse array of growth factors and physiologic stimuli in a PI3K–dependent manner. Activated Akt affects downstream markers such as 4E-BP1, p70-S6K, and PRAS40, and acts as a key mediator of signals for cell survival, proliferation, angiogenesis, and a number of metabolic effects of insulin.  

Eukaryotic initiation factor 4E binding protein 1 (4E-BP1) is an important effector of mTOR signaling. Hypophosphorylated 4E-BP1 acts as a translational repressor by binding and inhibiting the eIF4E.
Proline-rich Akt substrate of 40 kDa (PRAS40) is a direct substrate for AKT.

Invitrogen™ sandwich ELISA kits quickly detect and quantify site-specific phosphoproteins in cell lysates. Total kits normalize the protein content of the sample, independent of the phosphorylation state. ELISA kits allow for high throughput of samples in a 96-well format and get quantitative results reproducibly. Calibrated protein standards allow quantification in each experimental run. Using these tools together, you’ll gain a more detailed understanding of signaling proteins in the Akt pathway.

p70 ribosomal protein S6 kinase (p70-S6K) is a member of the ribosomal S6 kinase (RSK) family of serine/threonine kinases. The primary pathway by which most growth factors and cytokines activate p70-S6K appears to be the PI3K Akt-mTOR pathway.



HEK 293 cells were serum-starved overnight, then deprived of amino acids in PBS for 60 min. Cell extracts were prepared and analyzed with the 4E-BP1 [pT46] and 4E-BP1 (Total) ELISA. Compared to the untreated control, the Thr46 phosphorylation of 4E-BP1 is down-regulated in amino acid–starved HEK 293 cells, whereas total levels of 4E-BP1 remain relatively constant.


MCF-7 cells were serum-starved overnight, incubated with or without PI3K inhibitor wortmannin (PHZ1301) or mTOR inhibitor rapamycin (PHZ1233) then treated with IGF-I (PHG0074). Cell extracts were analyzed with the p70 S6K [pT389] ELISA and Invitrogen™ p70-S6K (Total) ELISA (KHO0571). Phosphorylation of p70-S6K is up-regulated in IGF-I–treated MCF 7 cells. This up-regulation is subject to wortmannin or rapamycin inhibition, whereas the total level of p70-S6K remains relatively constant.


Jurkat cells were treated with wortmannin at varying concentrations (50 to 500 nM) for 3 hr, and lysed and quantitated in parallel for total PRAS40 and PRAS40 [pT246] expression. The level of phosphorylation at Thr246 decreases with the dose of wortmannin. PRAS40 [pT246] levels were normalized to total PRAS40. Results correlate with western blots of the same samples (inset).


DescriptionSpecies
Size Cat. No.

Akt [pS473] ELISA Kit
Hu, Ms, Rt
96 tests
KHO0111
Order 
Akt (Total) ELISA Kit
Hu, Ms, Rt
96 tests
KHO0101Order
4E-BP1 [pT46]
Hu, Ms, Rt
96 tests
KHO0691Order
4E-BP1 (Total) ELISA Kit
Hu, Ms, Rt
96 tests
KHO0681Order
P70-S6K [pT389] ELISA Kit
Hu, Ms, Rt
96 tests
KHO0581Order
P70-S6K (Total) ELISA Kit
Hu, Ms, Rt
96 tests
KHO0571Order
PRAS40 [pT246] ELISA Kit
Hu
96 tests
KHO0421Order
PRAS40 (Total) ELISA Kit
Hu
96 tests
KHO0411Order

ABfinity™ Antibodies and the AKT Pathway

  • The next generation of antibodies
  • The most specific antibodies, for more reproducible results
  • Each lot tested with phosphorylation site-specific antibodies with peptide competition

ABfinity™ antibodies are the next generation of antibodies, exclusively from Invitrogen.  They are generated by cloning specific genes of antibodies, and then producing them in a mammalian expression system. With the help of ABfinity™ technology, we've generated the most specific antibodies to generate highly reproducible results.

Abnormalities in the PI3K/Akt cascading signaling pathway are associated with cancer, diabetes, cardiovascular conditions, neurological disorders, and other diseases. Developing effective treatments has required altering the activity of this pathway.
To help scientists looking at Akt signal transduction, we offer the Akt [pS473] ABfinity™ Recombinant Rabbit Monoclonal Antibody to study the effects of Akt cell signaling in glucose metabolism, cell cycle, cell survival, adhesion, and angiogenesis. All antibodies are tested against multiple species and with all major applications. In addition, we have the best portfolio of phosphorylation site-specific and total antibodies. Invitrogen is the only company to test each lot of phosphorylation site-specific antibodies with peptide competition, to make sure each lot detects only at the right site.

  • Learn More at www.invitrogen.com/pssa

 

 

Flow cytometry of Jurkat cells labeled with rabbit anti-Akt [pS473].
Jurkat cells were incubated with 50 µM LY294002 (red trace) or without (green trace) for 1 hr prior to being fixed and permeabilized using FIX&PERM® reagents. Cells were then stained with 1 μg Akt [pS473] followed by Alexa Fluor® 488 goat anti-rabbit IgG. Blue trace represents secondary antibody alone.
 
Immunocytochemistry of mouse fibroblasts cells labeled with rabbit anti-Akt [pS473].
Mouse fibroblast cells were treated with (top right) or without (top left) insulin and labeled with rabbit anti-Akt [pS473]. Signal is knocked down after incubation with the phosphopeptide used as an immunogen (bottom left), but not with the non-phosphopeptide (bottom right). Alexa Fluor® 488 goat anti-rabbit IgG was used as secondary antibody. Nuclei are stained with Hoechst (blue).

 

 

Immunohistochemistry of human esophagus carninoma tissue labeled with rabbit anti-Akt [pS473].
PE human esophagus carcinoma tissue was labeled with rabbit anti-Akt [pS473]. Tissues were pretreated with EDTA and detected with SuperPicTure™ Polymer DAB. Note nuclear and cytoplasmic staining in tumor cells.
 
Western blot of 3T3 lysates labeled with rabbit anti-Akt [pS473].
Rabbit anti-Akt [pS473] was used to label Akt [pS473] in untreated 3T3 lysates (lane 1) or PDGF-treated 3T3 lysates (lane 2).

 

DescriptionReactivity: Tested  (Predicted)ApplicationsSize Cat. No. 
PKC-θ [pT538] ABfinity™ Recombinant Rabbit Monoclonal AntibodyHu (X, Rt, Ms, Cp, B)WB,F, IHC, IF/ICC100 μg700043Order 
Mnk1 [pT197/pT202] ABfinity™ Recombinant Rabbit Monoclonal AntibodyHu (Z, X, Sw, Rt, P, Ms, Mk (Rh), Eq, Eq, Cp, Ch, Cn, B)WB,F, IF/ICC100 μg700242Order
Cul-2 ABfinity™ Recombinant Rabbit Monoclonal AntibodyHu, Rt, Ms (X, Or, Mk (Rh),  Eq, Cp, Cn, B)WB,F, IHC, IF/ICC100 μg700179Order
IRAK4 ABfinity™ Recombinant Rabbit Monoclonal AntibodyHu (Sw, Sh, Rt, Qu, Eq, Cn, B)F, IF/ICC100 μg700026Order
AMPKβ1 [pS182] ABfinity™ Recombinant Rabbit Monoclonal AntibodyHu (X, Rt, Or, Eq, Ch, Cn, B, Ms)WB,F, IHC, IF/ICC100 μg700241Order
Smad1/5 [pS463/pS465] ABfinity™ Recombinant Rabbit Monoclonal AntibodyHu (Z, X, Sw, Sh, Su, Rt, Ms, Mk (Rh), Eq, Cp, Ch, Cn, B)F, IHC, IF/ICC100 μg700047Order
4E-BP1 [pT37] ABfinity™ Recombinant Rabbit Monoclonal AntibodyHu (Z, Rt, Ms, B, Hu, Eq)E, WB,F, IHC, IF/ICC100 μg700238Order
STAT4 ABfinity™ Recombinant Rabbit Monoclonal AntibodyHu, Ms, Rt (Sw, Eq)E, WB,F, IHC, IF/ICC 100 μg700185Order
SUMO-3 ABfinity™ Recombinant Rabbit Monoclonal AntibodyHu, Ms, Rt (Mk (Rh), Cp)WB,F, IHC, IF/ICC100 μg700186Order
CASP3 [D175] ABfinity™ Recombinant Rabbit Monoclonal Antibody (clone 9H19L2)Hu (X, Sw, Sh, Rt, Rabbit, P, Ms, Eq, Ha, Fe, Eq, Cp, Cn, B)E, WB,F, IHC, IF/ICC100 μg700182Order

 

Live-Cell Imaging of Cell Cycle and Division

  • For the first time, real time—precise detection of cell cycle progression with ready-to-use fluorescent protein constructs; no need to purify plasmid or worry about vector integrity and quality
  • Titratable fluorescent proteins—prepackaged at a constant concentration; enable defined optimization due to the ability to precisely titrate expression levels
  • Cell-friendly—no lipids, dye-loading chemicals, or other potentially harmful treatments; primary and stem cells are labeled without apparent cytopathic effects

The Premo™ FUCCI cell cycle sensor enables live-cell imaging—as cells progress through the cell cycle, nuclear fluorescence changes from red to yellow to green.
In 2008, Miyawaki and colleagues developed the Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), a fluorescent protein (FP)–based sensor that employs red (RFP) and green (GFP) fluorescent proteins fused to different regulators of the cell cycle, Cdt1 and geminin. The Premo™ FUCCI Cell Cycle Sensor takes this technology one step further by using the BacMam gene delivery system—the prepackaged genetically-encoded reagents are ready for immediate use. Simply add the Premo™ FUCCI reagents to your cells, treat with the BacMam enhancer, wash, incubate overnight for protein expression, and visualize cell cycle progression using fluorescence microscopy.

Akt plays various roles in cell cycle progression.  With the Premo™ FUCCI Cell Cycle Sensor, researchers can accurately and easily visualize cell cycle progression in live cells.




Live-cell imaging with Premo™ FUCCI Cell Cycle Sensor.  U2OS cells treated were transduced with the Premo™ FUCCI Cell Cycle Sensor and co-stained with the far red-fluorescent Alexa Fluor® 647 wheat germ agglutinin.  Imaging was performed on live cells using a Delta Vision Core microscope and standard FITC/TRITC/Cy5 filter sets.