For the experiments, vector batches containing either IN-I-PpoI or IN-I-PpoIH78A were produced (Table 1) and pooled where needed.
SG carried out most of the experiments: vector construction, genetic transformation, selection and analysis of plants; WO coordinated some experiments (vector construction, analysis of expression) and took part in discussion of the project; ANO have made substantial contributions to conception and design, analysis and interpretation and wrote the manuscript.
WZ carried out most of the experiments: vector construction, Agrobacterium-mediated and biolistic transformation, selection and analysis of plants; WO coordinated vector construction and took part in discussion of the project; SG participated in vector construction and analysis; A N-O coordinated the work and wrote the manuscript.
For pull-down experiments, vectors encoding FLAG-tagged Krit1 and OSM were transfected into HEK293 cells (the American Type Culture Collection (ATCC) Rockville, MA, USA) using lipofectamine.
In parallel experiments, vector-only cells were generated to act as negative controls.
In control experiments, vector-transfected neurons migrated normally to the superficial layer (layers II IV) of the cortical plate (CP; Fig 4C).
One vector describes whether the gene is repressed and the other vector describes whether the gene is induced across of the microarray experiments; vectors were retained for analysis only if induction or repression was seen in at least 10% of experimental conditions (see Methods and Figure 2).
In contrast to the previous experiments, vectors used in this part of the study encoded the huC1 and huC2 linked to one another by the self-cleaving 23 amino-acid picornaviral 2A-like sequence from the porcine teschovirus-1 (P2A) to allow for efficient functional stoichiometric expression of multiple flanking proteins under the influence of the bone-specific col1a1 2.3 kB promoter.
In this experiment, a vector digested at a single site with various overhangs was added to cell free extract.
The pipeline takes as input raw Affimetrix CEL files and experiment design vector (or matrix in case of more complicated ANOVA statistics), which distinguish the biological replicates, time series etc. (e. g. sample versus control in the simplest case).
