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Fig. 3 | Molecular Brain

Fig. 3

From: Full UPF3B function is critical for neuronal differentiation of neural stem cells

Fig. 3

Mutations in UPF3B impair UPF3B protein NMD activity in neural stem cells. a. Expression of λN-HA-UPF3B proteins. Neural stem cells were transfected with pCI-λN-HA-UPF3B expression constructs and protein expression was analysed 48 h after transfection by 10 % SDS PAGE followed by detection by Western blotting. UPF3B proteins and α-tubulin were detected using anti-HA and anti-tubulin antibodies. b, c. Tethering assays. b. Neural stem cells were transfected with the control plasmid phRL-TK, the reference plasmid phrGFP-C and the various pCI-λN-HA-UPF3B expression constructs. 24 h after transfection, differentiation was induced. RNA was prepared 48 h after transfection and analysed by qPCR as described. c. Neural stem cells were transfected, treated and mRNA levels analysed as in B except that phRL-TK was replaced with phRL-TK-10BoxB. For the graphs, Renilla luciferase mRNA levels were standardised with respect to GFP mRNA levels, and Renilla luciferase mRNA levels in cells expressing UPF3B-Ala423 was defined as 1. The graphs show average Renilla luciferase mRNA levels from three independent experiments, error bars indicate the standard deviation. Asterisks indicate values significantly different from luciferase mRNA levels in the presence of UPF3B (one-way ANOVA followed by Dunnett's test; P < 0.05)

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