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.