Fig. 1. TRB3 ablation restores the p-AKT/p-mTOR axis in rd16 retinas.

A Images of western blot membranes treated with anti-p-4E-BP1, -4E-BP1, -p-mTOR, -mTOR, p-AKT^Ser473, -AKT, and -actin primary antibodies and secondary antibodies scanned with LI-COR imager in three groups of mice. B Quantitation of band density of western blot images demonstrates a statistically significant increase in p-4E-BP1 and p-AKT in rd16 TRB3^−/− retinas. Increase in p-4E-BP1 was also accompanied by an enhancement of mTOR activity (n = 4). C Images of a western blot membrane treated with anti-puromycin antibody to detect the translational level in retinas (n = 4). Retinal protein extracts containing incorporated puromycin as the result of intraperitoneal injection in mice were run to detect the density of incorporated puromycin. The density was normalized through protein loading detected by staining with coomassie blue (D). E Retinal protein extracts from P15 C57BL/6, rd16, rd16 TRB3^−/− mice were run on polyacrylamide gel. Western blot images of membranes immunoblotted with anti-Rhodopsin antibody are shown (n = 4). F Retinal cryosections of P20 C57BL/6, rd16, and rd16 TRB3^−/− mice were used to perform the IHC with antibody against rhodopsin (in green). Propidium iodide labels the nuclei of the outer nuclear (ONL) and the inner nuclear (INL) layer, and the retinal ganglion cells (RGC). Accumulation of rhodopsin in the ONL was observed in both rd16 and rd16 TRB3^−/− retinas. G Quantitation of the incorporated puromycin in animal retinas to detect translational level (C, D) and rhodopsin protein for western immunoblotting. E The restoration of the translational rate and an increase in rhodopsin level were observed in rd16 TRB3^−/− retinas (n = 4). Data are shown as mean ± SEM; a.u. = arbitrary units; the scale is 50 µm. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.