Protein Phosphorylation Site Mutants

pEGFP-HMW-MAP2 and pEGFP-NES-Jun were previously described (Björkblom et al., 2005 (link); Tararuk et al., 2006 (link)). Phosphorylation site mutants of HMW-MAP2, EGFP-HMW-MAP2^{T1619A/T1622A/T1625A} (abbreviated to GFP-MAP2-AAA) and EGFP-MAP2^{T1619D/T1622D/T1625D} (abbreviated to GFP-MAP2-DDD), were prepared by insertional overlapping PCR using mutagenic primers as previously described (Hongisto et al., 2008 (link)). The phosphorylation site numbering is based on the rat HMW-MAP2 Uniprot entry P15146. For in utero electroporation, the CMV promoter in EGFP-HMW-MAP2^WT and EGFP-HMW-MAP2^{T1619D/T1622D/T1625D} was changed to a CAG promoter for optimal expression in brain. MAP2C was isolated by PCR from rat brain cDNA. It was inserted downstream of GST in the pGEX-6P3 vector (GE Healthcare) using the pGEMTe cloning vector (Promega). pCDNA3-MKK7-JNK1 was a gift from Roger J. Davis (HHMI, Worcester, MA, USA).