Figure 6

Silencing of Thap1 in mouse primary striatal neurons leads to a decrease in the levels of the 32 kDa and 50 kDa T1-LIR species. (a) Quantitative real-time PCR assay of mTHAP1 was performed on cDNA derived from mRNA from untreated mouse primary striatal neurons and following transduction with lentiviral (LV) particles expressing mThap1, shThap21 or 96, or shLuc control sequences (left panel) (representative of 2 independent experiments) or AAV particles expressing a third shRNA sequence directed at mThap1 (N = 3) (Unpaired t-test: *p < 0.05; Untreated level arbitrarily set at 100%). (b) Left panel: Westem blotting of total cellular extract, 40 µg, from primary striatal neurons transduced with either LV-mThap1 or LV-GFP control alone or together with lentivirus carrying shRNA #96 sequence directed at mThap1 or non-silencing (NS) shRNA. Right panel: Identical experiment as in left panel except for replacement of LV with AAV with a different shRNA sequence, as in (a). MOI for LV = 2.5 viral particles/neuron; for AAV = 100,000 viral particles/neuron. (c) Graphs show densitometry of blots from experiments represented in (b). (N = 4 for LV-4, shRNA, N = 2 for AAV-shRNA). (NI = non-infected) (d) Transduction of primary striatal neurons with LV-mThap1 increases the intensity of the 32 (*) and upper 50 kDa (arrowhead) T1-LIR species in nuclear extracts (10 µg), and the relative increase is decreased by co-transduction with AAV-shRNA specific to Thap1 in the nucleus of primary striatum cultures.