Fig. 1

LZTR1 regulates the expression of four RAS GTPases, including MRAS, HRAS, NRAS, and KRAS. a HEK293 cells were transfected with control-siRNA pool or LZTR1-targeting-siRNA pool (LZTR1-siRNA pool). Twenty-four hours later, cells were cultured in serum-free or 10% serum-containing medium for an additional 24 h, and we evaluated the expression and activity levels of RAS and the RAS/MAPK signaling pathway by western blot. b, c The influence of LZTR1 overexpression on endogenous and exogenous RAS protein levels. HEK293 cells were seeded in 60 mm dishes and transfected with the indicated plasmids for 24 h, and cells were cultured in fresh medium for an additional 24 h. Then we evaluated the protein levels at 48 h after transfection. b Cells were transfected with 0, 0.5, 1, 2, 3, 4, 5, 6 µg LZTR1-pcDNA only. (c) Cells were transfected with Flagg-tagged RAS and LZTR1 (2.5:0 µg, 2.5:0.5 µg, 2.5:1 µg or 2.5: 2.5 µg ratios of RAS:LZTR1). d To perform the cycloheximide (CHX)-chase assay, each 2.5 µg Flag-RAS- and 1 µg Myc-LZTR1-expressing plasmid or empty plasmid was transfected into HEK293 cells for 48 h; then, cells were treated with 50 μg/mL cycloheximide (CHX). The cells were harvested at the indicated time points after treatment, and Flag-RAS expression levels were evaluated by western blot analyses. Band densities were analyzed using ImageJ software from the National Institutes of Health, and expression was normalized to ACTB protein levels