Drugs that disassemble microtubules promote ERM activation. (A) Schematic representation of MyrPB-ezrin-rLucII/rGFP-CAAX BRET2 biosensor (Leguay et al., 2021). (B) Diagram representing the identified class of targets of the validated ezrin activators. (C) BRET2 signals were measured in HEK293T cells expressing MyrPB-ezrin-rLucII/rGFP-CAAX and incubated with increasing concentration of nocodazole (blue) or podophyllotoxin (red) for 15 min. (D and E) Immunoblot of HEK293T (left) and HeLa (right) cells incubated with the indicated concentrations of nocodazole for 15 min (D). p-ERM over ERM signals were quantified and normalized to vehicle (DMSO; E). (F and G) Immunoblot of HEK293T cells incubated with 1 µM nocodazole for the indicated times (F). p-ERM over ERM signals were quantified and normalized to vehicle (t = 0; G). (H and I) Immunofluorescence of HEK293T cells incubated with vehicle (DMSO) or 1 µM nocodazole for 15 min (H). p-ERM staining at the plasma membrane was quantified and normalized to cells treated with vehicle (I; n > 45 cells). BRET2 signals (C) represent the mean ± SD of three independent experiments. Immunoblots (D and F) and immunofluorescences (H) are representative of at least two independent experiments. p-ERM quantifications represent the mean ± SD of at least two independent experiments. Dots represent independent experiments (E), individual cells (I), or the mean of independent experiments (C and G). P values were calculated using one-sample t test (E and G) or using two-tailed unpaired t test (I). *, P < 0.05; ****, P < 0.0001. ns, not significant. Numbers associated with Western blots indicate molecular weight in kD. Source data are available for this figure: SourceData F1.