Fig. 4
From: LZTR1 facilitates polyubiquitination and degradation of RAS-GTPases
The analysis of target Lys residues of RAS ubiquitinated by LZTR1. a A schematic diagram of the sequence alignments of HRAS, NRAS, KRAS, and MRAS. b In vivo ubiquitination assays were performed with MRAS-1KR mutants instead of wild-type. HEK293 cells were transfected with the indicated plasmids for 24 h followed by treatment with MG132 for 6 h. Thirty hours later, the polyubiquitination status was evaluated by western blot using anti-HA-tag, anti-Flag-tag, anti-Myc-tag, and anti-βactin antibodies. In (b), cells were seeded in 100 mm dishes and transfected with transfected with MRAS, Myc-LZTR1 and HA-Ub (10 µg:2 µg:7 µg ratio of RAS:LZTR1:Ub). c In vivo ubiquitination assays were carried out as in (b) using HRAS-WT and HRAS-K170R mutant. d HEK293 cells were transfected with HRAS-WT, HRAS-G12V, HRAS-K170R and HRAS-G12V/K170R in the presence or absence of Myc-LZTR1. Forty-eight hours later, we evaluated the influence of LZTR1 on HRAS levels. In (d), cells were seeded in 60 mm dishes and transfected with transfected with HRAS and Myc-LZTR1 (2.5 µg:0 µg or 2.5 µg:2.5 µg ratio of RAS:LZTR1)
