Neurofibromin

Frozen MEFs in culturing vessels or liquid nitrogen-frozen samples of adult brains were lysed in M-PER or T-PER protein extraction reagents containing protease and phosphatase inhibitors (all from Thermo Scientific, Waltham, MA). Protein samples (30 μg) were separated by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membrane. The membranes were then immunoblotted with indicated primary antibodies, followed by incubation with a horseradish peroxidase-coupled anti-rabbit IgG antibody (1:20,000; Jackson ImmunoResearch, West Grove, PA; 711-035-152). Protein bands were visualized with Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA). Primary antibodies used for immunoblotting were phospho ERK1/2 and ERK1/2 (1:5000; Cell Signaling Technology, Danvers, MA; 9101 and 9102), β-tubulin (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA; H-235) and neurofibromin (1:500; Santa Cruz Biotechnology; sc-67). Western signal band intensity was quantified using ImageJ Software (National Institutes of Health, Bethesda, MD).

Cell pellets, sciatic nerves, or tumors dissected from sciatic nerves were lysed in buffer containing 1% NP-40 (nonylphenoxypolyethoxylethanol) supplemented with protease inhibitors. Western blotting was performed as previously described [48 (link)]. Antibodies used included neurofibromin (Santa Cruz Biotechnology; dilution 1:100), p53 (Cell Signaling; dilution 1:1000), and α-tubulin (Sigma; dilution 1:10000). Densitometry was measured using Life Science Software from UVP VisionWorks LS Version 8.1.1 Image Acquisition and Analysis Software system. α-tubulin serves as an internal protein loading control.

Paraffin-embedded tonsil, 4 normal peripheral nerves (sural nerve), 23 NF1-DNFs (13 males, 10 females) and 55 sp-DNFs (31 males, 24 females) were included in the immunohistochemical analyses. All DNFs were incubated with neurofibromin (Santa Cruz sc-67) antibodies, while and all DNFs, tonsil and normal sural nerve sections were incubated with KIR2DL5 (Abcam; ab175895) antibodies. Biotinylated secondary rabbit antibodies (Vector Laboratories) were used in combination with Vectastain Elite ABC development and hematoxylin counterstaining. Normal human tonsil was used as the reference control tissue for the KIR2DL5 antibody. Images were acquired on a Nikon Eclipse E600 microscope conjugated with a Nikon Plan Fluor 10x/0.30 DIC L objective and a Leica EC3 camera. Normal human sural nerve, sp-DNF sections, as well as primary cell cultures of normal human Schwann cells and HEK293T cells were analyzed by immunofluorescence using S100β (Millipore CB1040), c-Kit (Millipore; MAB1164), tryptase (Abcam; ab2378), vimentin (DSHB; AMF-17b) and KIR2DL5 (Abcam; ab129751) antibodies conjugated to appropriate Alexa Fluor secondary antibodies. Images were acquired on a Nikon Eclipse TE300 fluorescence microscope conjugated with a Nikon Plan Fluor 20x/0.45 ELWD objective and a Leica DFC3000G camera.