Internalization and FRAP analysis of LET-2 and EMB-9 knock-ins. (A) Representative sum projections of confocal fluorescence z-stacks of LET-2::mNG (C) and EMB-9::mNG (C) at the L1, L2, L3, late L4, and gravid adult stages. Small aggregates in the body wall muscle were first detected in L3 larvae (47% of LET-2::mNG [C] and 73% of EMB-9::mNG [C], n > 10 each) and continued into gravid adult (100% for LET-2::mNG and EMB-9::mNG, n > 10 each). The scale bar is 20 μm. (B) Single z-slice fluorescence image of EMB-9::mCherry (IS2) muscle aggregates (magenta in merge) localized within the ER (visualized with ELO-1::mNG; cyan in merge) in L1 larvae. Yellow arrows point to aggregates in the ER (n = 10 animals; 10 aggregates counted per animal; 99/100 aggregates localized to the ER). The scale bar is 5 μm. (C) Left: Single z-slice images of LET-2::mNG (C), EMB-9::mNG (C), and EMB-9::mNG (IS1) prior to, immediately after, and 5 h after FRAP at the L1-proximal pharyngeal bulb. Yellow arrowheads point to photobleached regions. White arrows point to unbleached regions used as the control region for analysis. Dotted boxes indicate the location of magnified regions shown in the panel below. The scale bar is 1 μm for magnified panels. The scale bar is 10 μm for all other panels. Right: Boxplots show the percentage of fluorescent signal recovered after 5 h (n = 10 animals, P > 0.05, ANOVA with Tukey’s multiple comparisons).