Double-stranded oligonucleotide probes LY2109761 in vivo containing WT or mutant Pax6 binding sites ( Figure S6A) were radiolabeled. Pax6 polyclonal rabbit antibody (Covance) was used for supershifts. Competition assays used a 50- to 100-fold

molar excess of competitor probes in each binding reaction. Chromatin was extracted from 20 E12.5 mouse cortices. DNA-protein complexes were precipitated with anti-Pax6 antibody (Covance) or with anti-igG antibody (Abcam). ChIP was performed as described by Sansom et al. (2009). Primer pairs were selected to measure, by qPCR, the relative levels of fragments, each of which included one of the predicted binding sites (Figure S6B). Fragments around Cdk6 were isolated from a BAC clone (RP23-53B17) or genomic DNA using the primers listed in Table S2 and cloned into the pGL4.10 promoterless firefly luciferase reporter vector (Promega). Site-directed mutagenesis used the QuikChange Site-Directed Mutagenesis Kit (Stratagene) with the PCR primers listed in Table S3.

HEK293 cells were transfected using Lipofectamine 2000 (Invitrogen). Pax6 was expressed using the pCMV-Pax6 construct, generated by inserting full-length Pax6 cDNA into pCMV-Script plasmid (Stratagene). The Renilla luciferase vector was pRLSV40 (Promega). HEK293 cells were harvested 48 hr after transfections and analyzed with the Dual Luciferase R428 chemical structure Reporter Assay System (Promega). The primary antibodies were mouse anti-Pax6 (1:200; DSHB), rabbit anti-Pax6 (1:500; Covance), rabbit anti-Cdk6 (1:500; Santa Cruz), rabbit anti-cyclin D2 (1:200; Santa Cruz), mouse anti-pRb (1:200; BD PharMingen), rabbit anti-phospho-pRb (Ser780, 1:1,000; Cell Signaling), rabbit anti-phospho-pRb

(Ser807/811, 1:1,000; Cell Signaling), and rabbit anti-beta actin (1:2,000; Abcam). many Alexa-coupled secondary antibodies were used and blots were quantified using the Li-Cor scanning system. We thank Alan Ross for carrying out array hybridizations, Thorsten Forster for statistical analyses, Trudi Gillespie (IMPACT Facility) for help with imaging, Malgorzata (Gosia) Borkowska for doing the electroporations, and David Santamaria for the Cdk6 knockout mice, and Nicoletta Kessaris for the Emx1-creERT2 mice. This work was supported by the MRC and the Wellcome Trust. “
“Synaptic transmission initiates when an intracellular influx of Ca2+ triggers release of neurotransmitters from presynaptic nerve terminals, which is mediated by exocytosis of synaptic vesicles (Chua et al., 2010). Prior to exocytosis, a fraction of SVs is specifically docked to an electron dense region of the presynaptic plasma membrane, termed active zone, and activated by additional steps referred to as priming (Gray, 1963). During the past decade, major progress was made in unraveling the molecular composition of the exocytotic apparatus. Synaptic vesicles are among its best characterized components.