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. 2023 Apr 12;299(6):104698. doi: 10.1016/j.jbc.2023.104698

Figure 9.

Figure 9

DIRAS1 inhibits RAC1 and RAC1B binding to SmgGDS and reduces their prenylation. HEK293T cells were cotransfected with cDNAs encoding SmgGDS-558-HA and myc-tagged (A) RAC1 or (B) RAC1B along with increasing amounts of DIRAS1 cDNA and/or empty vector, such that all cells were transfected with equal amounts of total cDNA. HEK293T cells cotransfected with SmgGDS-607-HA and myc-tagged (D) RAC1 or (E) RAC1B cDNAs were also transfected with increasing amounts of DIRAS1 cDNA and/or empty vector in the same concentrations as in A and B. After 24 h, the cells were lysed and a portion of each lysate was saved for the total cell lysate. The remaining lysate was immunoprecipitated with the HA antibody, followed by ECL immunoblotting using HA and myc antibodies. To evaluate prenylation of RAC1 and RAC1B in the presence of DIRAS1, cells were transfected with myc-tagged RAC1 or RAC1B plus (C) SmgGDS-558 or (F) SmgGDS-607, in the presence or absence of cotransfected DIRAS1. Prenylation with the C15AlkOPP probe was measured as described in Figure 7. Results are representative of three independent experiments. Densitometry of the proteins detected in the immunoblots is shown in Fig. S3. ECL, ECL, enhanced chemiluminescence.