The glomerular fraction was isolated by using magnetic isolation by perfusion of Afatinib paramanetic beads. The procedure in brief is as follow. Animal was anesthetised by intraperitoneal injection of 2.5% avertin. Animal was then perfused with warm 40 ml PBS containing 8*109 magnetic beads into left ventricle after lancing the vena cava caudalis. Pressure was maintained at around 60 mm Hg through out the time of perfusion. Successful perfusion will turn the kidney and liver pale. After completion of the perfusion kidneys were harvested and minced into fine pieces. Kidney samples were then digested with collagenase A for 30 minutes at 37uC. Then the digested tissue was passed though 100 mm cell strainer on ice. Actin is the most prominent protein at synapses and abundant in presynaptic terminals as well as postsynaptic spines . Actin has been implicated in the organization, mobility, and exocytosis of synaptic vesicles . It regulates the mobility of membrane proteins, such as neurotransmitter receptors , and promotes anchoring of receptors via its coupling to scaffolding proteins of the postsynaptic density . Moreover, actin regulation is essential for activity-dependent morphological changes of dendritic spines. Thereby actin is linked to synaptic plasticity, learning, and memory . Whereas the importance of actin for various synaptic processes is well accepted, little is known about the mechanisms that control actin at synapses. Actin dynamics critically depend on the activity of ADF/cofilin that bind co-operatively to filamentous actin , accelerate the dissociation of actin subunits and sever F-actin . A tight regulation of ADF/cofilin activity is crucial for synaptic plasticity as shrinkage of dendritic spines requires active ADF/ cofilin , while spine enlargement and long-term potentiation depend on the inactivation of ADF/cofilin . A confined number of phosphatases and kinases, including LIM domaincontaining serine/threonine kinases , control ADF/ cofilin activity via dephosphorylation and phosphorylation of a conserved serine BAY-60-7550 manufacturer residue at position 3 . Dysregulation of the LIM kinase 1 -ADF/cofilin pathway in humans is thought to contribute to the synaptic defects found in Williams syndrome, a particular form of mental retardation, and autism spectrum disorders . Indeed, genetic ablation of LIMK1 decreased ADF/cofilin phosphorylation in the mouse brain and affects synaptic vesicle exocytosis, neurotransmitter release, spine morphology, postsynaptic plasticity, learning, and memory . Notably, inactivation of the ADF/cofilin family member n-cofilin disturbed spine morphology and postsynaptic plasticity, while presynaptic physiology was fully preserved . Thus, the molecule acting downstream of LIMK1 in presynaptic terminals remain unknown. Besides n-cofilin, also its close homolog ADF is present in the adult mouse brain .