Fluorescent Fusion Protein Visualization

Non-expanded (Q30) ataxin-1 and truncated ataxin-1 fusion proteins were constructed in pEYFP or pECFP vectors using the standard PCR and mutagenesis methods previously established in our laboratory (Menon et al., 2012 (link)). COS cells were grown in chamber slides in Dulbecco’s modified Eagle medium supplemented with 10% foetal bovine serum and 100 U/ml penicillin-streptomycin (Invitrogen Life Technologies). Cells were transfected with appropriate plasmid DNA using GeneCellin tranfection reagent (BioCellChallenge). Cells were fixed using 4% paraformaldehyde 54 h post-transfection and slides were mounted using CitiFluor (Agar Scientific). Cells were observed and recorded using a laser scanning confocal microscope (de Chiara et al., 2009 (link)).