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.