MATERIALS AND METHODS
Cell culture: HEK293 (WT or NF1 +/+) cells were obtained
from ATCC (CRL-1573) and cultured in DMEM + 10% FBS and 1X Pen/Strep
using standard culture procedures. NF1 -/- or null HEK293 cells
were previously created through CRISPR Cas9 targeting NF1 exon 2
(Wallis et al., 2018) . We have chosen to evaluate functional NF1
expression and Ras activity in HEK293 cells because this cell line is
well characterized, used historically in NF1 research, easily takes up
exogenous DNA, and is easy to culture and scale. HEK293s have all three
Ras isoforms and recapitulate Ras signaling; hence, HEK293 cells are an
appropriate model system for these assays.
Nf1 cDNA plasmid development: The wild type (WT)Nf1 cDNA plasmid was developed by GeneCopoeia and is commercially
available. The full-length mouse cDNA (mNf1) produces a
>250 kDa neurofibromin protein that is capable of
modulating Ras signaling (Wallis et al., 2018). We created an empty
vector (EV) control plasmid with the same parental backbone that does
not contain the Nf1 cDNA. We also created a panel of mutant cDNAs
representing variants found in NF1-affected individuals with different
clinically relevant phenotypes and assessed their ability to produce
mature neurofibromin and restore Nf1 activity inNF1 -/- cells. mNf1 cDNA is appropriate
for study because the full-length cDNA sequences of endogenoushNF1 and mNf1 have 92% sequence identity; amino acid
sequences share 98% identity and human cDNAs have historically been
unstable and toxic. Variants were introduced into shuttle vectors by
either site-directed mutagenesis or utilization of synthetic DNA
fragments and then cloned into the full-length vector using standard
enzymes. Each variant plasmid was confirmed by sequencing the entireNf1 cDNA insert and all subsequent DNA preparations were
validated by spot checking for the variant of interest. Furthermore,
multiple plasmid preps were utilized for each variant to mitigate any
variability due to quality of DNA.
Transient transections: For Western blots and GTP-Ras assays,
cells were transfected using LipoD293 (SignaGen Lab. Cat# SL100668)
with up to 1ug of cDNA per 6-well dish seeded with 500,000 cells per
well. Assays were performed 48-72 hours later. For titration
experiments, WT cDNA was balanced with EV control such that 1000 ng
total DNA was transfected.
Western blotting: Cells were lysed with RIPA buffer
supplemented with a protease inhibitor cocktail and phosSTOP, and
lysates were cleared by centrifugation at 20,000 RPM for 20 minutes at
40C. Protein was quantified with a Bradford assay and
50 ug of protein was loaded per well for NF1 blots and 10 ug of protein
was loaded for other blots. 8% SDS-polyacrylamide gels were run at 100
V for 2 hours and transferred at 100 V for 2 hours onto PVDF. Blots were
probed overnight at 40C with primary antibody, washed,
and probed for 1 hour at room temperature with secondary. Primary
antibodies include N-Terminal NF1 (Cell Signaling cat# D7R7D 1:1000),
tubulin (Abcam cat# ab52866 1:1000), b-actin (Cell Signaling cat# 3700
1:1000), p-ERK (Cell Signaling cat# 9101 1:1000), and total ERK (Cell
Signaling cat# 9102 1:1000). Secondary was HRP tagged from Santa Cruz.
Chemiluminescent substrate from Bio-Rad was used as per manufacturer’s
protocols.
RAS-G-LISA Assay: The RAS-G-LISA assay was obtained from
Cytoskeleton Inc. and was performed according to the manufacturer’s
instructions.
Statistical analysi s. All assays were repeated a minimum of
three times for each variant. For each experiment wild type Nf1cDNA (WT) and empty vector (EV) plasmid control with no cDNA insert were
included as controls. Depending on whether we intended to show either 1)
presence of WT cDNA repressed Ras activity more than absence ofNF1 cDNA (EV) as in our titration experiments or 2) variant cDNA
expression or activity was different from WT cDNA, we normalized
expression or activity to WT or EV respectively and made comparisons
with EV or WT respectively. Normalization to one control allows us to
combine data across independent experiments, and combination of multiple
repeat experiments controls for the effects of differential transfection
efficiencies. Statistical comparisons using student’s t-test were made
using Excel software to determine which results were statistically
significant.