Fig. 4

Grk5l Limits mTOR Signaling

(A) Western blot analysis of zebrafish embryos (6–8 ss) for S6K1 phosphorylation. Representative images of a single blot are shown. Bar graph displays means ± SEM.

(B) Western blot of mouse heart lysates of WT and GRK5 KO mice for phosphorylation of S6K1 and its target ribosomal protein S6. Bar graph displays means ± SEM.

(C) Overexpression of Grk5l-GFP in cells attenuates TORC1 activity toward S6K1. Bar graph displays means ± SEM.

(D) Cell volume of Grk5l transfected cells (left: mean ± SEM). Cells were controlled for grk5l expression (right graph: means ± SD, n = 2–3).

(E) Reversal of the Grk5l lateralization phenotype by rapa. Analysis of spaw in the presence of DMSO or rapa from 6 to 10 hpf. CTRL MO^DMSO versus Grk5l MO^DMSO: p < 0.0001; Grk5l MO^DMSO versus Grk5l MO^Rapa: p = 0.0033.

(F) Inhibition of S6K1 activity by PF-470871 reverses spaw misexpression upon Grk5l KD. CTRL MO^DMSO versus Grk5l MO2^DMSO: p = 0.0063; Grk5l MO2^DMSO versus Grk5l MO2^PF: p < 0.0001.

(G) Coimmunoprecipitation of transfected GFP or hemagglutinin (HA)-Grk5l-GFP and endogenous Raptor using an HA antibody. IP, immunoprecipitation; WCL, whole-cell lysate.

(H) Relative expression levels of grk5l obtained by qPCR (mean ± SD, n = 2).

(I) raptor mRNA levels (mean ± SD, n = 2).

See also Figures S2, S3, and S4.