Figure 2.

Semiquantitative reverse-transcription PCR, qRT-PCR and Western blot validation confirming on a selection of differentially expressed genes identified by microarray. (a) Semiquantitative reverse-transcription PCR validation: Fgf7 is upregulated in Ube3a^m−/p+ mice, whereas the rest of the genes, including Glra1, Mc1r, Nr4a2, Epha6 and Slc5a, are confirmed to be downregulated. NTC: no template control. (b) qRT-PCR validation showing the normalized mean fold change from the biological triplicates. The fold change is calculated using 2^{−(mean wild-type ΔCT−mean Ube3a (m−/p+) ΔCT)}; ‘+' and ‘−' represent upregulation and downregulation of transcript, respectively; ^*P<0.05. (c and d) Total protein (10 μg) extracted from mouse cerebellum was analyzed by SDS–PAGE using 6% acrylamide gel. Western blot analyses using antibody against Mc1r and Nr4a2 show that the 35 kDa Mc1r (c) and the 66 kDa Nr4a2 (d) proteins, respectively, are downregulated in the Ube3a^m−/p+ mice. β-Actin is used as endogenous internal control in the Western blot analyses.