miRNA mimics transfections and western blot

Chemically synthesized, double-stranded RNAs mimicking mature endogenous miRNAs were used for transfection into HeLa cells (ATCC). 100,000 cells per well of a 12-well plate were seeded one day prior transfection. 30 picomoles per well of miRNA mimics (Syn-hsa-miR-370-5p miScript miRNA Mimic and Syn-hsa-miR-543 miScript miRNA Mimic, both from Qiagen) were transfected using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. 48 hours after transfection cells were harvested and lysed in SDS PAGE Sample buffer 2x (25mM EDTA, 100mM Tris, 20% Glycerol, 4% SDS, 2% 2-Mercaptoethanol, 0.004% Bromophenol blue). Mouse brain lysates were kindly provided by Dr. C. Cemal (Imperial College of Science, Technology and Medicine, London, UK) and dissolved in SDS PAGE Sample buffer 2x. Samples were boiled for 5 min at 95°C and proteins were analyzed on 10% SDS gels and blotted onto PVDF-membranes (Roche). Blots were blocked in milk and incubated with the following antibodies: ATXN3 (1:1000; 986, [16]), DNAJB1, (1:1000; Cell Signalling 2118L), GAPDH (1:5000; Cell Signalling, 2118L).

The resulting bands were quantified using AIDA software. Statistical analyses were performed using one-way ANOVA with post hoc Dunnett’s test to accommodate for multiple comparisons or Student’s TTest for two-group comparisons, as appropriate.