Extended Data Fig. 6: Cell fractionations for plasma membrane (PM), nuclear envelope (NE), and cytoplasmic (C) fractions. The RAS:RanGAP1 complexes are present in many tumor types and non-tumorigenic lines.
a NCI-H23 cells were fractionated for PM, NE, and cytoplasmic fractions whose purity was verified by specific marker proteins, for example, EGFR and CD44 for the PM, lamin A/C for the NE, and α-tubulin for the cytoplasmic marker protein. KRAS is present in all three fractions, RanGAP1 is present only in NE and cytoplasmic fractions, while BRAF is present only in PM and cytoplasmic fractions. b-i Lysates from indicated fractions were IP with antibodies to BRAF, KRAS, RanGAP1, or mock IgG, followed by IB with antibodies to KRAS, BRAF, or RanGAP1. WCE, whole cell extract, Input, indicated purified fraction. KRAS formed a complex with BRAF in the WCE (b), PM (c,d), and cytoplasmic fraction (e). KRAS formed a complex with RanGAP1 in cytoplasmic fraction (f) and NE (g). h As BRAF is not present in NE (a), there was no complex formation between KRAS and BRAF in NE fraction. i Since RanGAP1 is not present in PM (a), there was no complex formation between KRAS and RanGAP1 in PM fraction. j-p Lysates from indicated samples were IP with antibodies to KRAS, RanGAP1, or mock IgG, followed by IB with antibodies to RanGAP1 or KRAS. WCE, whole cell extract. KRAS:RanGAP1 protein complexes were identified in: PDX’s from pancreas adenocarcinoma (j), PDX’s from colon adenocarcinoma (k), and PDX’s from HRAS mutant Nasopharyngeal Carcinoma (l), PDX’s from NRAS mutant colon adenocarcinoma (m), non-transformed HBEC line (n), non-immortalized, non-transformed WI-38 fibroblasts (o). In contrast to the positive results of complex formation between KRAS and RanGAP1 in primary human lung cancer shown in Fig. 5 l,m, the complex formation was not detected between RanGAP1 and the RAP1 (p). Two independent experiments were performed for each image, with similar results.
