Figure 2

Figure 2

Nebulin expression is reduced in neb mutants. (A) Nebulin transcript levels, determined from the RNAseq data, were drastically reduced in the nonsense and neb^hu28 mutants, whereas the levels in the splice mutants were consistent with those in the wt siblings (numbers indicate the padj values). (B) SDS-agarose gel stained with Coomassie blue showing reduced levels of nebulin in all mutants. (C) Confocal micrographs of myofibers stained with antibodies against Neb-N and α-Actinin. The nonsense, neb²¹, and neb^hu28 mutants show altered localization and drastically reduced levels of nebulin. The localization pattern of nebulin in neb³⁴ and neb³⁰ splice mutants is similar to that in the wt myofibers; however, nebulin levels are significantly reduced. To better illustrate the altered localization of nebulin, the brightness in the region marked by the square in left column panels was increased to 150% in panels showing high magnification detail of Neb-N staining. Scale bars = 10 μm. (D) Quantification of fluorescence intensity of anti-Neb-N antibody staining in myofibers (mean ± SEM). Violin plot showing that fluorescence intensity is significantly reduced in neb³⁴ and neb³⁰ mutants compared to wt (56.5% in neb³⁴ and 92.7% in neb³⁰, respectively). n = 56 measurements/genotype. Asterisks indicate p-values (** ≤ 0.01; **** ≤ 0.0001). See Supplemental Table 1 for fluorescence intensity value measurements and statistics.