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. 2016 May 25;36(21):5808–5819. doi: 10.1523/JNEUROSCI.3857-15.2016

Figure 2.

Figure 2.

PSMD13-mediated ubiquitination-dependent degradation of misfolded R91W RPE65. A, Immunoblot analysis of RPE65 and PSMD13 in RPE of WT and KI eyecups transfected with the indicated amounts of PSMD13 siRNA (siPSMD13) or negative control siRNA (siCont, 200 nm). The 26S proteasome non-ATPase regulatory subunit 11 (PSMD11) and LRAT were detected as internal maker for the 19S cap of the proteasome and RPE, respectively. B, C, Relative immunoblot intensities of RPE65 and PSMD13 normalized to LRAT were expressed as fold of RPE65 (B) or percentage of PSMD13 (C) in eyecups transfected with control siRNA. D, Confocal microscopic images of RPE cells in 129S2/Sv mice eyecups transfected with pcDNA or pcDNA encoding EGFP. Nuclei were stained with DAPI. E, Chymotrypsin-like and trypsin-like activities of the proteasomes in RPE of WT and KI mice eyecups transfected with 200 nm PSMD13 siRNA or control siRNA. F, Immunoblot analysis of RPE65, PSMD13, and the indicated proteins in WT and KI eyecups transfected with pRK5 or pRK5 encoding PSMD13. G, H, Relative immunoblot intensities of RPE65 and PSMD13 normalized to LRAT in F. I, Chymotrypsin-like and trypsin-like activities of the proteasomes in RPE of WT and KI mice eyecups transfected with pRK5 or PSMD13 construct (pPSMD13). J, RPE65 in WT and KI eyecups transfected with siPSMD13 or siCont was immunoprecipitated and probed with an Ub antibody. K, Immunoblot analysis of RPE65 and PSMD13 in RPE of WT and KI eyecups maintained at 37°C or 30°C after transfecting pRK5 or pPSMD13. L, Relative immunoblot intensities of RPE65 and PSMD13 (bottom) in the KI mice eyecups in K. Asterisks indicate statistically significant differences between test and control groups (*p < 0.05, **p < 0.005). Error bars indicate SD (n = 4).