for in utero electroporation , timed pregnant e13.5 cd1 mice were used ( the morning of the vaginal plug was e0.5 ) . one l of purified dna ( 1 - 2g/l ) mixed with 0.005% fast green was injected in utero into the lateral ventricle and electroporated into the neocortical ventricular zone according to published protocols26 . brains were pfa - fixed and vibratome - sectioned for analyses by confocal microscopy . in order to determine the spatial interaction between pten and myosinva , pc12 cells were transfected with p - mcherry - mvag and/or pten - egfp - c2 , differentiated for 2 days with ngf ( 50 ng / ml ) and fixed for flim experiments to measure frster resonance energy transfer fret . time - domain flim experiments and data analysis were performed as described previously16 . the globular domain from chick myosinva ( mvag ) was amplified by pcr ( proofstart;qiagen ) ( primers : 5-gccaccatgagtctgcagcatgagatcacc-3 and 5-gacacgtgatatgaaacccag-3 ) and cloned into pca-ires - egfpm5-grip , p - mcherry n1 ( clontech ) or p3xflag - cmv-7.1 ( sigma ) . similarly , mouse cdna was used to clone the globular domain of myosinvb ( primers : 5- atgtccaccatcaacggcattaag-3 and ttgacttcattgagaaactccag-3 ) and myosinvc ( primers : 5-atgagtgctattaacggcatcaagc-3 and 5- ttcagtctccggagaaagccg-3 ) into p3xflag - cmv-7.1 . site directed mutagenesis of parental flag - mvag or mvag - iresgfp vectors were performed using the quikchange ii xl mutagenesis kit ( stratagene ) according to the manufacturer s protocols . the primers used were 5-gggtgaaaccaacagggctgcaaaacataacatccagcattgctgatga-3 and 5-tcatcagcaatgctggatgttatgttttgcagccctgttggtttcaccc-3 for the mva rkrqni mutation , and 5-gctgcacagttgttgcaagtgcaaaacataacagatgaagatgca-3 and 5-gcttctgcatcttcatctgttatgttttgcacttgcaacaactgt-3 for the mva cortices or livers from e18 rat embryos were homogenized in lysis buffer ( 50 mm tris ph 7.5 ; 150 mm nacl ; 1% triton x-100 ; 2 m naov and 500 m naf ) containing protease inhibitors ( roche ) . immunoprecipitations were performed overnight ( 4c ) using 5g of anti - pten antibody ( a2b1 , santa cruz ) or mouse control antibody and protein - g beads ( pierce ) . after washing , beads were boiled in loading buffer and separated on 10% sds - polyacrylamide gels , which were fixed and stained using brilliant blue g - colloidal concentrate ( sigma ) according to manufacturer s instructions . selected protein bands were isolated , processed and sequenced by proteome sciences plc . in short , after in gel trypsin digestion , samples were analysed by lc / ms / ms , the mass spectral data was processed into peak lists and searched against the swiss prot and ncbi non - redundant databases using mascot software ( matrix science , uk ) . for co - immunoprecipitation experiments , hek293 cells were transfected with different constructs using lipofectamine 2000 and anti - flag m2 affinity gel ( sigma ) was used for isolating the 3xflag - mvag complex . western blot analysis of immunoprecipitates was performed using anti - flag ( sigma ) , anti - gfp ( abcam ) or anti - pten antibodies ( santa cruz or cell signaling ) . cortices from e15.5 dilute lethal and wildtype littermates were used to isolate total rna ( qiagen rneasy mini kit ) , which was reversed transcribed into random primed cdna ( reverse transcription kit , promega ) and served as template for all qpcr experiments . myosinva , myosinvb , myosinvc and pten mrna levels were measured relative to gapdh , pgk1 and hprt . primers for qpcr analysis of the different mrnas were ordered from qiagen ( quantitect primer assays ) , quantifast sybr green ( qiagen ) was used as a reporter for detection with the light cycler1.0 ( roche ) . for in utero electroporation , timed pregnant e13.5 cd1 mice were used ( the morning of the vaginal plug was e0.5 ) . one l of purified dna ( 1 - 2g/l ) mixed with 0.005% fast green was injected in utero into the lateral ventricle and electroporated into the neocortical ventricular zone according to published protocols26 . in order to determine the spatial interaction between pten and myosinva , pc12 cells were transfected with p - mcherry - mvag and/or pten - egfp - c2 , differentiated for 2 days with ngf ( 50 ng / ml ) and fixed for flim experiments to measure frster resonance energy transfer fret . the globular domain from chick myosinva ( mvag ) was amplified by pcr ( proofstart;qiagen ) ( primers : 5-gccaccatgagtctgcagcatgagatcacc-3 and 5-gacacgtgatatgaaacccag-3 ) and cloned into pca-ires - egfpm5-grip , p - mcherry n1 ( clontech ) or p3xflag - cmv-7.1 ( sigma ) . similarly , mouse cdna was used to clone the globular domain of myosinvb ( primers : 5- atgtccaccatcaacggcattaag-3 and ttgacttcattgagaaactccag-3 ) and myosinvc ( primers : 5-atgagtgctattaacggcatcaagc-3 and 5- ttcagtctccggagaaagccg-3 ) into p3xflag - cmv-7.1 . site directed mutagenesis of parental flag - mvag or mvag - iresgfp vectors were performed using the quikchange ii xl mutagenesis kit ( stratagene ) according to the manufacturer s protocols . the primers used were 5-gggtgaaaccaacagggctgcaaaacataacatccagcattgctgatga-3 and 5-tcatcagcaatgctggatgttatgttttgcagccctgttggtttcaccc-3 for the mva rkrqni mutation , and 5-gctgcacagttgttgcaagtgcaaaacataacagatgaagatgca-3 and 5-gcttctgcatcttcatctgttatgttttgcacttgcaacaactgt-3 for the mva cortices or livers from e18 rat embryos were homogenized in lysis buffer ( 50 mm tris ph 7.5 ; 150 mm nacl ; 1% triton x-100 ; 2 m naov and 500 m naf ) containing protease inhibitors ( roche ) . immunoprecipitations were performed overnight ( 4c ) using 5g of anti - pten antibody ( a2b1 , santa cruz ) or mouse control antibody and protein - g beads ( pierce ) . after washing , beads were boiled in loading buffer and separated on 10% sds - polyacrylamide gels , which were fixed and stained using brilliant blue g - colloidal concentrate ( sigma ) according to manufacturer s instructions . selected protein bands were isolated , processed and sequenced by proteome sciences plc . in short , after in gel trypsin digestion , samples were analysed by lc / ms / ms , the mass spectral data was processed into peak lists and searched against the swiss prot and ncbi non - redundant databases using mascot software ( matrix science , uk ) . for co - immunoprecipitation experiments , hek293 cells were transfected with different constructs using lipofectamine 2000 and anti - flag m2 affinity gel ( sigma ) was used for isolating the 3xflag - mvag complex . western blot analysis of immunoprecipitates was performed using anti - flag ( sigma ) , anti - gfp ( abcam ) or anti - pten antibodies ( santa cruz or cell signaling ) . cortices from e15.5 dilute lethal and wildtype littermates were used to isolate total rna ( qiagen rneasy mini kit ) , which was reversed transcribed into random primed cdna ( reverse transcription kit , promega ) and served as template for all qpcr experiments . myosinva , myosinvb , myosinvc and pten mrna levels were measured relative to gapdh , pgk1 and hprt . primers for qpcr analysis of the different mrnas were ordered from qiagen ( quantitect primer assays ) , quantifast sybr green ( qiagen ) was used as a reporter for detection with the light cycler1.0 ( roche ) .