Figure 3
FKBP12 interacts with mHTT and increases higher-order and low A11-reactive oligomeric mHTT species in N2A cells.
(a) Htt(Q)25-eYFP and Htt(Q)109-eYFP (co-expressed with FKBP12 or vector alone) were immunoprecipitated (IP) with V5 antibody and probed with 1C2 antibody in the absence/presence of DSP (left and middle panels). Western blot was performed to examine expression of Htt(Q)25-eYFP, Htt(Q)109-eYFP and FKBP12-V5 using the indicated antibodies (right panel). (b) Cell lysates from each transfectants were fractionated using size exclusion column to determine the sizing behavior of HTT proteins. Each column fraction was analyzed by SDS-PAGE/Western blot with 1C2 or V5 antibodies. The positions of the molecular mass standards are depicted using arrowheads. (c,d) Fractions 7–11 of the Htt(Q)25-eYFP, Htt(Q)25-eYFP/FKBP12, Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12 samples were probed with 1C2 and quantified using Image J. (e,f) Non-denatured fractions 7–11 from Htt(Q)109-eYFP and Htt(Q)109-eYFP/FKBP12 samples were applied onto a slot-blot apparatus, probed with anti-oligomer A11 antibody and quantified using Image J. Bars represent means ± SEM (n = 4). ***p < 0.001, as analyzed by Student’s t-test.
