Western blot analysis of extracts from ZR-75-1 cells, untreated or λ phosphatase and CIP-treated, using Phospho-SSH3 (Ser37) Antibody (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Western blot analysis of extracts from various cell lines, untreated or Calyculin A #9902-treated, using Phospho-SSH3 (Ser37) Antibody (upper) or α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
Western blot analysis of extracts from 293T cells, untransfected or transfected with a construct expressing Myc/DDK-tagged full-length human SSH3 protein, using Phospho-SSH3 (Ser37) Antibody (upper) and Myc-Tag (71D10) Rabbit mAb #2278 (lower).
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised June 2016
Protocol Id: 263
Phospho-SSH3 (Ser37) Antibody recognizes endogenous levels of SSH3 protein only when phosphorylated at Ser37.Species Reactivity:
Polyclonal antibodies are produced by immunizing animals with a synthetic phosphopeptide corresponding to residues surrounding Ser37 of human SSH3 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Slingshot homolog 3 (SSH3) is a protein phosphatase that modifies actin cytoskeleton dynamics via cofilin dephosphorylation. Cofilin is an evolutionarily conserved, actin-binding protein that severs actin filaments during processes that rely on actin filament dynamics, including cytokinesis, cell migration, invasion, and neuronal development. Actin severing and filament depolymerization are regulated through the controlled cycling of cofilin between the phosphorylated and dephosphorylated forms (1). The kinases LIMK and TESK inactivate cofilin by phosphorylating it at Ser3 (2,3). The slingshot homologs (SSH1, SSH2, and, to a lesser extent, SSH3) and chronophin/PDXP phosphatases remove phosphate from cofilin at Ser3, enabling cofilin binding to actin and filament depolymerization (3). SSH3 is widely expressed in epithelial tissues, and has been found to be non-essential for viability and fertility in knockout mice (4). While its biological function remains elusive, phosphorylation at Ser37 of SSH3 has been identified in several phosphoproteomic studies (5-7).
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