Constructs of GFP (green fluorescent protein) were made with polyglutamine (82Q and 19Q) and binding proteins (TBP and CBP); yellow fusion protein was bound to molecular chaperone Hsp70 and CFP.
HeLa and 023 cells were co-transfected (Liptofectamine, Invitrogen) with 82Q-Flag or Htt-150Q (Huntingtin)and the GFP constucts. The localization of protein aggregates with observed throughout the cytosol and nuclear area by fluorescent microscopy using primary antibodies anti-Flag antibody (M5, 1:500, Sigma) or polyclonal HP-1 which recognizes aa 80-113 of Huntingtin protein (gift of M. MacDonald, Harvard Univ.) and detected with TnTC-conjugated secondary antibodies (red color). Filter sets for GFP and Texas Red were used on a Zeiss epifluorescent microscope and Nikon epifluorescence scope. Adobe Photoshop 5.5 was used for pseudocoloration and merging of the images.
FRAP (fluorescence recovery after photobleaching)and FLIP (fluorescence loss in photobleaching) studies were performed on 023 transfected cells for fluorescent intensity, mobile fraction and diffusion coefficient.Images were collected at various times and observed for recovery or loss. Graphpad Prism software (Graphpad, San Diego) was used.
FRET analysis used co-transfected HeLa cells with either CFP or YFP fusion constructs. Fluorescent energy transfer relys on a donor (CFP) emission and the acceptor (YFP). FRET signal images were analysed on 20 regions of interest using Metamorph imaging software.