Fig 3

Fig 3

(A) Phosphorylated ribosomal protein S6 kinase 1 (S6K1) (upper panel) and total S6K1 (lower panel) were detected in protein lysates of whole atp6v1e1b-deficient zebrafish and their corresponding WT controls at 4 dpf by immunoblotting. Asterisks indicate non-related, cross-reactive bands. Representative immunoblots are shown. We confirmed equal loading by staining for α-tubulin. (B-D-F) Band intensities of chemiluminescent signals of non-phosphorylated proteins were quantified with ImageJ and normalized to WT controls. (C-E-G) Band intensities of chemiluminescent signals of phosphorylated proteins were quantified with ImageJ. The ratio of phospho-p85-S6K to total p85-S6K, phospho-p70-S6K to total p70-S6K, and phospho-p31-S6K to total p31-S6K was normalized to WT controls. Data are expressed as mean ± SD from 3 biological replicates in B-G. (H-P) Gene expression of Notch-target genes (her4, her6, and hey2), Wnt-target genes (axin2, tcf7, and lef1), and AMPK-target genes (prkaa1, ptch1, and gli1) were investigated by RT-qPCR. Data are expressed as mean ± SD from 5 biological replicates in H-P.