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. 2023 Jun 16;26(7):107157. doi: 10.1016/j.isci.2023.107157

Figure 3.

Figure 3

DIRAS3 interacts with RAC1 and stimulates its polyubiquitination

(A) Calu-1 cells were transfected with Flag-DIRAS3 or control plasmid and cultured for 24 h before further incubating with MG132 (20 μM) or E64D (15 μM) for 6 h. Cell lysates were analyzed by western blotting with antibodies against Flag, RAC1, and ACTB.

(B) Co-immunoprecipitation analysis of the ubiquitination of RAC1 in 293FT cells co-transfected with Flag-DIRAS3 plasmid, Myc-RAC1 plasmid, and HA-Ub plasmid.

(C) 293FT cells were transfected with Myc-RAC1 and Flag-DIRAS3 plasmids and were cultured for 24 h before being further incubated with MG132 (20 μM) for 6 h. Then, the co-immunoprecipitation assays were carried out with Flag antibody and the co-eluted proteins were detected by western blot assays with Flag and Myc antibodies.

(D) Co-immunoprecipitation of endogenous RAC1 with DIRAS3 in H1299 cells.

(E) Immunofluorescence staining for detecting endogenous DIRAS3 and RAC1 expression in Calu-1 cells. The intensity profiles of DIRAS3 and RAC1 along the white line are plotted in the right panel.