Fig. 4

Huntingtin is located in the cytosolic fraction of lymphoblast cells

(A) Detection of huntingtin. A Western blot of proteins in subcellular fractions of lymphoblasts obtained from a normal individual (N) and an HD homozygote (HD) was probed with a 1:10,000 dilution of HF1. Fractions are denoted by PN, postnuclear supernatant; S, 100,000 × g supernatant (S100); P, 100,000 × g pellet (P100), and N, nucleosol. The position of the 200-kD marker (myosin) is shown, and the positions of the ∼350-kD normal (N) and HD (HD) huntingtin are indicated by arrows. The bands at ∼240 and ∼260 kD are also detected by the preimmune sera. (B) Detection of control proteins. Western blots of proteins in the subcellular fractions of lymphoblasts shown in Panel A were probed with control antibodies. The top panel shows the results obtained using a monoclonal antibody directed at pRB, a 110-kD nuclear protein that is detected in the N fraction (24), and the bottom panel the results with a monoclonal antibody directed at pERK1 (Transduction Laboratories), a 44-kD plasma membrane associated protein that is detected in the P fraction (25). The band at ∼45 kD is probably a phosphorylated form of ERK1 that is also recognized by this monoclonal. In separate experiments (not shown), similar Western blots were probed with polyclonal rabbit antisera against ornithine aminotransferase (OAT), a mitochondrial protein (26) that was detected in the P fraction and neurofibromin (27), a cytoplasmic protein that can associate with microtubules that was found in both the P and S fractions (28).