Figure 7

Mutational and functional status of key genes in 2XSB cells. (a) Summary of mutations identified by SNP array and/or WES sequencing analyses in genes previously implicated in MPNSTs and/or included in the Bushman Laboratory Cancer Gene List. Gene names that are underlined represent likely pathogenic calls and all others are pathogenic mutations. For copy number changes, the numbers in parentheses indicate the copy number of each haplotype, while the numbers outside the parentheses indicate the total copy number. If a mutation was not identified in a gene, the box in the mutation column is gray. (b) Immunoblot blot analysis of cell line-derived whole cell lysates from 2XSB cells and a control cell line (293 T). Lysates were prepared from 2D cultures of log phase cells and were probed with anti-C-terminal NF1, anti-N-terminal NF1, anti-CDKN2A (p16), and anti-p53 antibodies. Actinin was used as a loading control. (c) Ras activation assays were performed to determine the functional status of NF1 in serum deprived 2XSB cells stimulated with 10% serum for 5 min. Activated Ras was identified by Raf pull-down (Raf PD) with the Raf-Ras binding domain beads followed by immunoblotting with pan-Ras and NF1 antibodies. Activation of downstream effectors of Ras was determined in whole cell lysates by immunoblot analyses with anti-pCRAF and -pERK antibodies. Lysates were also probed with anti-total ERK and pan-Ras antibodies. GAPDH was used as a loading control.