Fig. 3

LZTR1 homodimerizes with itself and forms a complex with RAS and CUL3. a Cellular localization of Myc-LZTR1 and Flag-RAS in HEK293 cells. The cells were transfected with the indicated expression plasmids and stained with anti-Myc-tag (red), anti-Flag-tag (green), and NucBlue Stain (nuclei, blue). Cells were seeded at 5 × 10⁴ cells/well, grown on 13 mm2 glass coverslips in 24-well plate for 24 h, and then transfected with indicated plasmids. The ratio of RAS and LZTR1 was 5:1 (1 µg: 0.2 µg) and we evaluated 30 h after transfection. The yellow arrows indicate representative locations where LZTR1 and RAS overlap. b Cells were seeded in 100 mm dishes, transfected with with Flag-KRAS-pCAGGS (10 µg), Myc-LZTR1-pcDNA (4 µg), and CUL3-V5-pcDNA (4 µg) for 30 h. The lysates were subjected to immunoprecipitation assays with Flag-M2 agarose, and then we evaluated their interactions by western blot analyses using anti-Flag-tag, anti-Myc-tag, anti-V5, and anti-βactin antibodies. c A schematic diagram of the domain organization of LZTR1. d–f HEK293 cells were transfected with the indicated expression plasmids, and the lysates were subjected to co-immunoprecipitation assays. The immunoprecipitants were subjected to western blot analyses using anti-Flag-tag, anti-Myc-tag, and anti-βactin antibodies. In (d), cells were transfected with 7.5 µg Flag-KRAS-pCAGGS and 7.5 µg Flag-LZTR1 domains for 48 h. In (e), cells were transfected with 7.5 µg Flag-CUL3-pcDNA and 7.5 µg Myc-LZTR1-domain-pcDNAs for 48 h. In (f), cells were transfected with 7.5 µg Flag-LZTR1-pcDNA and 7.5 µg Myc-LZTR1-pcDNAs for 48 h. g A schematic diagram of the homodimerized-LZTR1/RAS/CUL3 complex