Technical Reference #1735

1735.

Activated Actin-Depolymerizing Factor/Cofilin Sequesters Phosphorylated Microtubule-Associated Protein during the Assembly of Alzheimer-Like Neuritic Cytoskeletal Striations Ineka T. Whiteman; Othon L. Gervasio; Karen M. Cullen; Gilles J. Guillemin; Erica V. Jeong; Paul K. Witting; Shane T. Antao; Laurie S. Minamide; James R. Bamburg; Claire Goldsbury, University of Sydney, Journal of Neuroscience, 29(1735), (2009)
Link To Paper

Materials & Methods:
Antibodies and reagents. Mouse monoclonal antibodies are actin (1A4; Dako) -actin (Abcam) (III)-tubulin (Abcam) tau phosphorylated at Ser202/Thr205 (AT8; Pierce) and Ser262/356 (12E8; Elan) (Seubert et al. 1995). The monoclonal antibody 12E8 raised against Ser262-phosphorylated tau is known to crossreact with other phosphorylated MAPs such MAP2c MAP4 (Timm et al. 2003) and doublecortin (Schaar et al. 2004) since these MAPs also contain the 12E8-specific KXGS motifs in their microtubule-binding domains. However 12E8 has strong affinity for tau in which the microtubule-binding domain contains several KXGS motifs the target sequence for 12E8 (Seubert et al. 1995; Yoshida and Goedert 2002; Timm et al. 2003; Schaar et al. 2004). Rabbit polyclonal antibodies are ADF (1439) ADF (D8815; Sigma-Aldrich) Ser3- phosphorylated ADF/cofilin (Shaw et al. 2004) cofilin (C8736; Sigma-Aldrich) actin (A2066; Sigma-Aldrich) and total tau (A0024; Dako). Phalloidin 488 (Invitrogen) was used to visualize F-actin (Invitrogen). Anti-mouse and anti-rabbit secondary antibodies included Alexa Fluor (488 555 and 647)-conjugated (Invitrogen) for immunofluorescence and horseradish peroxidase-conjugated (GE Healthcare) for immunoblotting. Lyophilized amyloid peptides were from Bachem.

Microscopic Technique
Epifluorescence and Immunofluorescence Microscopy

Cell Type(s)
Neurons