Fig. 4: AFF4 is a P70S6K substrate.

A Insulin-regulated phosphosite mapping of AFF4 and domain annotation with zoomed insert of multiple sequence alignment indicating the Akt/P70S6K motif surrounding human S836 (rodent S831 and zebrafish S771). B Phosphorylation of S831 on AFF4 from 10-week versus 73-week-old mouse bone (n = 3 biological replicates/group, *q < 0.1; limma moderated t-test with Benjamini Hochberg FDR). C–D Western-blot analysis of HEK293 lysates (n = 3 biological replicates) for known insulin-regulated phosphorylation events in the presence of an Akt or P70S6K inhibitor. Quantification of mouse S831 phosphorylation on AFF4 by LC-MS/MS is also shown. E P70S6K in vitro kinase assay on purified mouse AFF4 by LC-MS/MS (n = 2 biological replicates/condition, *p < 0.01, unpaired two-sided t-test). F–G CRISPR/Cas9 double knockdown of rps6kb1/2 in zebrafish or scramble negative control (scr)(scale bar = 1 mm) followed by assessment of growth and mineralization via Alizarin red staining (***p < 0.001, unpaired two-sided t-test), (H) and western-blot analysis of tailfin P70S6K protein levels and phosphorylation of S235/6 on RPS6. Phosphoproteomics of CRISPR/Cas9 double knockdown (n = 8 biological replicates/condition; each replicant a pool of n = 15 individual fins) of rps6kb1/2 in zebrafish or scramble negative control (scr) with (I) volcano plot showing the phosphopeptide Log2 fold-change (rps6kb1/2 versus scr) against the -Log10 p-value and (J) highlighting the phosphorylation of S771 on AFF4 and S236/40 on RPS6 (*p < 0.05, **p < 0.01, unpaired two-sided t-test with permutation-based FDR). Data are represented as +/− standard error of the mean. Source data are provided as a Source Data file.