Constructs, antibodies and cell-lines
NF1 minigene exon trap constructs and NF expression plasmids were generated using standard cloning techniques [Sambrook et al., 1989], Gibson assembly [Gibson et al., 2009] and/or site-directed mutagenesis (see Supplementary Information for details). All constructs were verified by sequencing of the complete insert. Nucleotide and amino acid numbering are according to NF1 transcript NM_000267.3 and SPRED1 transcript NM_152594.2, unless specified otherwise.
Antibodies were from Cell Signaling Technology (Danvers, U.S.A.)(rabbit anti-HA; mouse anti-HA; 9B11 mouse anti-myc), Invitrogen (mouse anti-V5) Sigma-Aldrich (St. Louis, U.S.A.) (mouse and rabbit anti-FLAG) and Li-Cor Biosciences (Lincoln, U.S.A.)(goat anti-rabbit 680 nm and goat anti-mouse 800 nm conjugates). Anti-FLAG affinity beads were from Sigma-Aldrich, glutathione-sepharose was from GE Healthcare (Uppsala, Sweden).
HEK 293T and COS-7 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM)(Lonza, Verviers, Belgium) containing 10% fetal calf serum, 50 U/ml penicillin and 50 μg/ml streptomycin in a humidified 37oC, 5 - 10% CO2 incubator.