Western blot analysis of extracts from A431 cells, untreated or EGF-treated (100 ng/ml for 5 minutes), using Phospho-Afadin (Ser1718) Antibody.

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Western Blotting Protocol

A. Solutions and Reagents

B. Protein Blotting

A general protocol for sample preparation.

C. Membrane Blocking and Antibody Incubations

I. Membrane Blocking

II. Primary Antibody Incubation

D. Detection of Proteins

Directions for Use:

* Avoid repeated exposure to skin.

posted June 2005

revised November 2013

Western Blot Reprobing Protocol

B. Protocol

revised October 2016

Protocol Id: 10

Product Usage Information

Specificity / Sensitivity

Phospho-Afadin (Ser1718) Antibody detects endogenous levels of l-afadin protein only when phosphorylated at serine 1718.

Species Reactivity:

Species predicted to react based on 100% sequence homology:

Mouse, Rat, Monkey, Dog

Source / Purification

Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser1718 of human afadin. Antibodies are purified using protein A and peptide affinity chromatography.

In multicellular organisms, intercellular junctions play essential roles in tissue integrity and maintenance of cell polarity. Tight junctions (TJs) form a continuous barrier to fluids across the epithelium and endothelium (reviewed in 1). Adherens junctions (AJs) are dynamic structures that form cell-cell contacts linking cells into a continuous sheet (reviewed in 2). The actin filament-binding protein, Afadin, binds to nectin forming a connection to the actin cytoskeleton (3). AJs are formed when nectin assembles cadherin at the cell-cell adhesion site and these junctions are then involved in the formation and maintenance of TJs (4,5). Afadin has two splice variants: l-afadin, which is ubiquitously expressed, and s-afadin, which is expressed predominantly in neural tissue. s-Afadin is a shorter form lacking one of the three proline-rich regions found in l-afadin, as well as the carboxyl-terminal F-actin binding region (6). Human s-afadin is identical to AF-6, the ALL-1 fusion partner involved in acute myeloid leukemias (7). Recent work has also shown that afadin is involved in controlling the directionality of cell movement when it is localized at the leading edge of moving cells (8,9).

Phospho-Afadin (Ser1718) Antibody is directed at a site that was identified at Cell Signaling Technology (CST) using PhosphoScan®, CST's LC-MS/MS platform for modification site discovery. Phosphorylation at Ser1718 was discovered using an Akt substrate antibody. Please visit PhosphoSitePlus®, CST's modification site knowledgebase, at www.phosphosite.org for more information.

Entrez-Gene Id

Swiss-Prot Acc.

Data Sheets & Documentation Data sheet (85KB)

For Research Use Only. Not For Use In Diagnostic Procedures.

Upstream / Downstream Explore pathways related to this product.

Upstream / Downstream Proteins

STRING - Known and Predicted Protein-Protein Interactions.

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