Fig 1: PSMC5 mediates Tln1 ubiquitination and degradation.(A) HUVEC lysates were incubated with in vitro-synthesized biotin-labelled sense or antisense AZIN2-sv for biotin pulldown followed by immunoblot analysis. (B) RIP assays were performed using an antibody against PSMC5 or negative control IgG. The purified RNA was used for qRT-PCR analysis, and the enrichment of AZIN2-sv was normalized against the input. ?p < .05 vs. RIP with IgG; n = 6 per group. (C) Agarose gel electrophoresis of RIP products. Arrow indicates the enriched AZIN2-sv. (D) PSMC5 protein levels detected by western blotting when AZIN2-sv was overexpressed and knocked down (GAPDH is the internal reference). (E) HUVECs with AZIN2-sv overexpression or knockdown were treated with MG132 (5 µM) for 24 h. Cell lysates were immunoprecipitated with an antibody against Tln1 or PSMC5. The precipitates and input were analysed by immunoblotting. (F) Immunoblots for PSMC5 and Tln1 in HUVECs transfected with PSMC5-targeting siRNAs or scrambled control siRNAs (GAPDH is the internal reference). ?p < .05 vs. control; n = 6 per group. (G) HUVECs transfected with PSMC5-targeting siRNAs or scrambled control siRNAs were treated with MG132 (5 µM) for 24 h. Cells lysates were immunoprecipitated with either control IgG or antibody against Tln1. The precipitates and input were analysed by immunoblotting.
Fig 2: Ninety minutes following the retrieval session, synaptic fractions were obtained in order to analyze the phosphorylation of the Rpt6 subunit using western blots. In the BLA (A), 22-mo old males display reduced pRpt6 (B), while females show no difference (C). However, in the mPFC (D), while males show no differences in pRpt6 (E), females 22-mo old females display reduced pRpt6 (F). Results are expressed as mean percentages of 3-mo old rats within each sex (± SEM). Blots have been cropped for clarity to show representative samples. Here, the mPFC is defined as the area including both the prelimbic cortex (PL) and infralimbic cortex (IL). *Indicates P < 0.05.
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