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. 2017 Sep 25;114(41):E8760–E8769. doi: 10.1073/pnas.1620153114

Fig. 1.

Fig. 1.

K63-polyUb is present at excitatory synapses and targets PSD-95 as a substrate. (A) Immunoblot (IB) analysis of K63-polyUb species in forebrain lysates prepared from mice at different postnatal stages. (B) Total ubiquitin and K63-polyUb (after stripping and reprobing) species in mouse forebrains (P30) and cultured rat hippocampal neurons at 21 d in vitro (DIV21) after immunoprecipitation (IP) with an anti-ubiquitin antibody. (C) Immunostaining of K63-polyUb and PSD-95 in cultured rat hippocampal neurons. [Scale bars, 20 μm (Upper) and 5 μm (Lower).] Arrowheads denote colocalized PSD-95 and K63-polyUb clusters, and arrows indicate K63-polyUb clusters lacking PSD-95 costaining. (D) Correlation plot of PSD-95 and K63-polyUb cluster intensities in C. Pearson’s r is shown. (E) K63-polyubiquitination of endogenous PSD-95 in mouse brain. WCL, whole-cell lysate. (F) K63-polyubiquitination of PSD-95–Flag in transfected HEK293FT cells. (G) Schematic showing ubiquitin lysine mutants used in this study. (H) Effects of HA-ubiquitin and mutants in promoting K63 ubiquitination of PSD-95–Flag in HEK293FT cells. Experiments were performed in the absence of proteasome inhibitors.