Figure S3 from LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors
Year: 2023
Type: other
Abstract: Supplementary Figure 3. (A) Shown are western blots using antibodies directed against T202/Y204 phosphorylated ERK1/2 following incubation with a range of concentrations of LXH254 over a 4 - 72 hour period. Which individual RAF protein is not expressed in each HCT 116 variant is indicated above the relevant panels. (B) Shown are western blots using Abs directed against the indicated proteins in either whole cell lysates (WCL) or material immuno-precipitated from cell lysates using Abs directed against ARAF (ARAF IP) or BRAF (BRAF IP). Lysates were generated from either parental MIA PaCa-2 cells or a MIA PaCa-2 variant lacking expression of CRAF. (C) Recombinant MEK1 can be phosphorylated by immuno-precipitates from both LXH254 treated and untreated MIA PaCa-2 and MEL-JUSO cells using an anti-body directed against ARAF contains MEK1 kinase activity. Proteins detected in either whole cell lysates (WCL) or ARAF-Ab immuno-precipitates (IP:ARAF) are indicated on the right. (D) IP-kinase activity on recombinant MEK1 in immuno-precipitates from HEK293 cells expressing FLAG-tagged ARAF using Abs directed against FLAG requires ATP and occurs on both wild type (WT) and kinase-dead (K97M) MEK1. (E) Shown are western blots using antibodies directed against T202/Y204 phosphorylated ERK1/2 following incubation with a range of concentrations of LXH254 over a 4-72 hour period. The individual RAF protein expressed in each HCT 116 variant is indicated above the relevant panels. (F) Shown are western blots of phosphorylated MEK1/2 (S218/S221) and ERK1/2 (T202/Y204) following a 4 hr. incubation with a range of LXH254 concentrations in the RAS/RAF wild type cell line PC-9.
Authors: Kelli-Ann Monaco, Scott Delach, Jing Yuan, Yuji Mishina, Paul Fordjour, Emma Labrot, Daniel McKay, Ribo Guo, Stacy Higgins, Hui Qin Wang, Jinsheng Liang, Karen Bui, John Green, Peter Aspesi, Jessi Ambrose, Felipa Mapa, Lesley Griner, Mariela Jaskelioff, John Fuller, Kenneth Crawford, Gwynn Pardee, Stephania Widger, Peter S. Hammerman, Jeffrey A. Engelman, Darrin D. Stuart, Vesselina G. Cooke, Giordano Caponigro
Language: English
Topic: Melanoma and MAPK Pathways
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4361890435