Abolishing RHO-1 binding of ANI-1 prevents cortical enrichment of the C-terminus. (A) Single z-plane GFP-tagged ANI-1 variants treated with ani-1(RNAi). A zoom-in of the equatorial region for the indicated time points is shown on the right. Since selected ANI-1 mutants exhibited reduced cortical accumulation in comparison to GFP::ANI-1^WT, the scaling of their fluorescence intensity was increased as indicated. Scale bars are 5 µm. (B) Embryonic lethality in % for the indicated transgenes and RNAi conditions. The number of progenies (n) counted is indicated. (C) Normalized cortical fluorescence intensity of GFP::ANI-1 variants from the anterior (0%) to the posterior (100%) pole at indicated time points. (D) Mean normalized fluorescence intensity of GFP::ANI-1 variants at the cell equator for indicated conditions at 180 s after NEBD. (E) Immunoblot of indicated GFP::ANI-1 and FLAG::ANI-1 expressing adult hermaphrodites probed with anti-GFP (left) or anti-FLAG (right) and anti-Actin antibodies. The NG::ANI-1 is an in-situ tagged ANI-1 harboring also a FLAG tag. The membrane on the left was cut and the upper part was treated with the anti-GFP and the lower part with the anti-Actin antibodies. Since the FLAG::ANI-1^C-term and actin bands were very close to each other, the membrane on the right was first treated with anti-FLAG antibodies and imaged. Afterward, the membrane was washed with buffer and incubated with anti-Actin antibodies. For all, time in seconds (s) after NEBD is indicated, error bars are SEM. P values were calculated using two-tailed Student’s t test or Mann–Whitney U test and are n.s. P > 0.05, **P < 0.01, and ***P < 0.001. Source data are available for this figure: SourceData FS4.