BioPath Online™

New pathway web pages
Whether your Integrin pathway research involves basic research tools, cell-based assays, or comprehensive screening services, Invitrogen has solutions for you. See our new Integrin pathway web page for more information.

New Integrin pathway web page Empower your research today using Invitrogen’s comprehensive portfolio of products and services to investigate the Integrin pathway—everything from high-quality reagents for basic research and assay development to validated biochemical and cell-based assays, and world-class profiling and screening services. See our portfolio of Integrin pathway–associated reagents at www.invitrogen.com/integrin.

Integrins form the major family of proteins that mediate cell adhesion to other cells and the extracellular matrix. Intregrins not only perform key cell adhesion functions, but also propagate the signaling cascade through classic signaling pathways.

These signaling pathways affect the most basic of cell functions, such as proliferation, apoptosis, differentiation, and mobility. 
Malfunction of these signaling pathways are known in the cases of many tumors.  Many types of tumors can overexpress or unexpress different integrins, resulting in malfunctioning cells and the cellular ability to metastasize.
   
Integrin signaling involves signals from the extracellular matrix or other cells, resulting in many fundamental changes in cellular functions. This signaling involves integrin binding subsequent to the assembly of a focal adhesion plaque. This focal adhesion plaque includes molecules like, FAK, tensin, α-actinin, vinculin, talin, and paxillin.

For many cells, proliferation requires adhesion to the extracellular matrix,through integrins. If the integrins are attached correctly to the extracellular matrix, they can cascade the signal through focal adhesion plaques, resulting in phosphorylation or dephosphorlation of many proteins, including retinoblastoma protein, E2F, Cyclin A, P21, and P27.

Different types of integrins also inhibit or induce apoptosis and differentiation by their interaction to different extracellular matrices. Once again focal adhesion plaques, mediated by phosphorylation of molecules such as FAK, Ras-ERK, and Src play a major role in these fundamental cellular functions.

To study these signaling pathways, Invitrogen offers over 4,000 antibodies, with new antibodies released every month:

• Over 400 are phosphorylation site specific antibodies, developed to detect only phosphorylated proteins.
• Each lot manufactured with patented, ISO control methods and tested with peptide blocking to make sure only the highest quality, highly specific antibodies are produced.
• These antibodies are an essential tool in the study of integrins. 

		Figure 1. Extracts of serum-starved mitotic HeLa cells generated by treatment with 100 ng/ml taxol for 16 hours were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF.
Figure 2. Visualizing focal adhesion kinase with FAK [pY397] PSSA. HeLa cells were fixed and permeabilized using ice-cold 95% methanol. FAK [pY397] PSSA was labeled with Alexa Fluor® 488 dye (green fluorescence, Molecular Probes® Zenon® Alexa Fluor® Rabbit IgG Labeling Kit, Cat. no. Z25302), and nuclei were counterstained with DAPI (blue fluorescence, Cat. no. D21490). The image was captured using a Zeiss Axioplan 2 fluorescence microscope fitted with a 100× oil immersion lens.

Integrins play an important role in cell attachment to other cells or to the extracellular matrix, as well as in signal transduction. Focal Adhesion Kinase (FAK) is a non receptor protein tyrosine kinase that localizes to focal adhesions. In response to integrin engagement, FAK is autophosphorylated at tyrosine residue 397. This event creates high affinity binding sites for the SH2 domains of several important signaling proteins, including Src family members.

FAK is under investigation in several areas of research, including cell adhesion and migration studies, studies on migration in response to growth factors, and differentiation, cell cycle, apoptosis, and cancer studies.

Another protein with an interesting integrin signaling function is β-catenin, which is a component in the adherens junctions of epithelial cells. It binds to the cytoplasmic tails of different cadherins, forming a stabilizing interaction.

Figures 1 and 2. 3T3L1 cells were treated with 1 mM sodium orthovanadate for 5 hrs. and untreated 3T3L1 cells were used as control. Cell extracts were prepared in 0.1% SDS extraction buffer and analyzed using the FAK [pY397] ELISA and FAK (Total) ELISA. The FAK [pY397] detected phosphorylated FAK recombinant protein and phosphorylated FAK in orthovanadate treated 3T3L1 cells, but not non phosphorylated FAK recombinant protein or non-phosphorylated FAK in untreated 3T3L1 cells (Figure 1). In contrast, FAK (Total) ELISA kit detected both phosphorylated and non phosphorylated FAK recombinant protein and FAK in orthovanadate treated cells and untreated control (Figure 2).
Figure 3. Treatment with PP1, a specific inhibitor of Src kinase, inhibited phosphorylation of Src in Colo 201 cells. This inhibition occurred in a dose-dependent manner as assayed by Src[pY418] ELISA. Colo 201 cells were treated with PP1 at varying concentrations for 3 hrs., lysed, and assayed in parallel for both Src [total] amount and Src [pY418]. The amount of Src remained comparable, while the levels of phosphorylation at tyrosine 418 decreased with increasing doses of PP1.
	Figure 4. The data presented show that the β-Catenin ELISA kit detects β-catenin protein in cell lysates from human GTL16, Jurkat, HeLa, MCF-7, and mouse NIH3T3 cells. Levels of β-catenin protein detected with this ELISA kit are consistent with results obtained by Western blot analysis.

Invitrogen offers phosphoELISA™ kits for both phosphorylation site-specific proteins and total proteins to elucidate specific target proteins cell signaling pathway.

Cat. No.	Description	Quantity	Price
KHO0441	FAK [PY397] ELISA KIT	96 tests	Inquire
KHO0431	FAK (TOTAL) ELISA KIT	96 tests	Inquire
KHO0171	HU SRC [PY418] ELISA KIT	96 tests	Inquire
KHO0161	HU SRC (TOTAL) ELISA KIT	96 tests	Inquire
KHO1211	HU B-catenin (TOTAL) ELISA KIT	96 tests	Coming soon! Notify me when available

• Please visit www.invitrogen.com/pELISA to see the complete menu

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LanthaScreen™ kinase activity assays have been developed and validated for several key kinases in the integrin pathway.

LanthaScreen™ assays provide users the ability to rapidly apply TR-FRET technology to detect kinase activity in a simple and purified system where kinase, fluorescein-labeled substrate, and ATP are allowed to react:

• Following the kinase reaction, EDTA (to stop the reaction) and terbium-labeled antibody are added to each well.
• The terbium-labeled antibody binds to the phosphorylated fluorescein substrate, resulting in an increased TR-FRET value.
• The TR-FRET value is calculated as the ratio of the acceptor (fluorescein) signal to the donor (terbium) signal.

The amount of antibody that is bound to the tracer is directly proportional to the amount of phosphorylated substrate present; in this manner, kinase activity can be measured by an increase in TR-FRET value.

LanthaScreen™ Cellular Assays provide a proximal readout to analyze the phosphorylation and/or other post-translational modification status of key pathway proteins in living cells:

• This system utilizes cell lines that stably express GFP-fusion proteins (for example, HEK293E cells expressing GFP-AKT).
• The phosphorylation state of GFP-fusion is then analyzed in cell lysates using a terbium-labeled phosphospecific antibody in a time-resolved Föerster-resonance energy transfer (TR-FRET)-based readout (for example, phosphorylation of AKT at Thr308 by PDK1 is detected using Tb-anti-AKT [pThr308] antibody).

Measurement of PAK activity using Lanthascreen™ technology. Protein kinases within the integrin signaling pathway can be assayed using the LanthaScreen™ reagent toolbox.  Two-fold serial dilutions of kinase (PAK1(PV3820; in black), PAK4(PV4212; in blue), or PAK7 (PV4405; in red)) were incubated with 200 nM fluorescein-crosstide substrate (PV3509) and 1mM ATP in a 384-well plate. A 2X solution of 20 mM EDTA and 4 nM LanthaScreen™ Tb-pCrosstide Antibody (PV3564) was added to stop the reaction and develop the TR-FRET signal (left panel). Inhibition of PAK4 (34 nM) and PAK7 (30 nM) at 1 M ATP was performed with staurosporine (middle panel) and purvalanol A (right panel). Purvalanol A is a known weak inhibitor of the PAK kinases.





Measurement of PDK1-mediated AKT phosphorylation using the Lanthascreen™ AKT Cellular Assay. LanthaScreen™ AKT HEK293E cells (K1615) were plated in 32 l of assay medium. On the day of the assay, cells were either first treated with 4 μl of 1% DMSO followed by 4 μl of 10X concentration of IGF-1 (dose-response) for 30 min (top panel), or pretreated with the indicated amount of BX-795 (PDK1 inhibitor) or PI-103 (PI3K/mTOR dual inhibitor) prior to stimulation with IGF-1 (bottom panel). Cells were lysed by adding 20 μl LanthaScreen™ Cellular Assay Lysis Buffer (PV5598), which included 5 nM of Tb-anti-AKT [pT308] antibody (PV5584) and both protease/phosphatase inhibitor cocktails.

• Learn more about LanthaScreen™ Cellular Assays