The association of spectrin with cell membrane requires the PH domain of β-Spec, but not its Ank-binding domain. All images are z-projections at lateral position. (A and A’) A Triton X-100–permeabilized pupal eye disc containing GFP-positive β-spec^C mutant MARCM clones was stained for phalloidin. Note the decreased cortical F-actin level in β-spec^C mutant PECs. (B) Normalized mean phalloidin membrane intensity in A, C”, D”, E”, and F”. Data are means ± SEM (n ≥ 20 cells, representative of five animals). ****, P < 0.0001. N.S., no significance. (C–F’”) Triton X-100–permeabilized pupal eye discs containing GFP-positive MARCM clones of the indicated genotypes were stained for Myc (epitope on the β-Spec transgenes) and phalloidin. Note the failure of β-SpecΔPH to localize to cell membrane and its inability to rescue cortical F-actin level in β-spec^C mutant PECs (C–C’”). By contrast, Myr-β-SpecΔPH (D–D’”), wild-type β-Spec (E–E’”), or β-Spec^α8 (F–F’”) accumulated on cell membrane and rescued the cortical F-actin level in β-spec^C mutant PECs. Quantification of phalloidin membrane intensity is shown in B. Scale bars, 5 µm.