Fig. 6

mTORC1-induced developmental acceleration of the retina requires an immunoproteasome subunit Psmb9. a The 20S proteasomes were purified from E14.5 Tsc1-het and Tsc1-cko littermate mouse retinas by 20S Proteasome Purification Kit^® (Enzo Life Sciences), and caspase-like (β1/β1i), trypsin-like (β2/β2i), and chymotrypsin-like (β5/β5i) activities of the purified proteasomes were analyzed by measuring fluorescent intensities produced by cleaved peptide substrates Z-LLE-AMC (β1/β1i), Bz-VGF-AMC (β2/β2i), and Scu-LLVY-AMC (β5/β5i) (see details in Methods). The values are averages and error bars denote SD (n = 3; 3 independent litters). b Relative mRNA levels of 20S proteasome subunits in E14.5 Tsc1-cko and rapamycin-treated mouse retinas were obtained by comparing real-time quantitative PCR (RT-qPCR) values with those of Tsc1-het and vehicle-treated mouse retinas at the same age. The values are averages obtained by 6 (Tsc1-cko/Tsc1-het) and 7 (rapamycin/vehicle) independent measurements with mRNA isolated from 4 (Tsc1-cko/Tsc1-het) and 6 (rapamycin/vehicle) independent batches. c Relative levels of 20S proteasome subunit proteins in the mouse retinas were also examined by WB with corresponding antibodies. d Image pixels of WB bands in Tsc1-cko samples were calculated by the ImageJ software and relative intensities were obtained by comparing the pixel numbers with those Tsc1-het samples. Values are average measurements of 4 independent WB results. e Incorporation of Psmb9 into the proteasome was examined by WB detection of Psmb9 in 20S proteasome core complex, which was isolated by the 20S Proteasome Purification Kit. P-values are obtained by Student’s t-test and shown in the graphs (*<0.05; **<0.01)