LET-2/EMB-9 BM ratios are not precisely 2:1 and differ between tissues. (A) Top: a schematic diagram of a late L4 larva with analyzed tissues and surrounding BMs (dashed lines) shown. Bottom: single z-slice confocal fluorescence images showing LET-2::mNG (C) and EMB-9::mNG (C) in tissues analyzed. Yellow arrows indicate BMs measured (see Materials and methods). The scale bar is 20 μm. (B) Top: boxplot of the mean BM fluorescence intensity measurements of LET-2::mNG (C) normalized to the average of all mean BM fluorescence intensity measurements of EMB-9::mNG (C) for the same tissue and trial (see Materials and methods, n > 10 animals for each of three trials, ****P > 0.0001, ns, not significant, Brown–Forsythe ANOVA tests followed by Dunnett’s T3 multiple comparisons test). Colors of the data points indicate different trials. Bottom: table showing the ratios of the average BM fluorescence levels of LET-2::mNG (C)/EMB-9::mNG (C) for each tissue and trial. Colors in the table correspond to data points in the boxplot. Ratios at the spermathecal BM were consistently over 2 (LET-2::mNG [C] levels were more than double EMB-9::mNG [C] levels), while ratios at other BMs were consistently under 2. (C) Table showing what the composite ratio of LET-2/EMB-9 BM protein levels suggests for trimer diversity and the tissues where those ratios were found.