Fig. 1
Antisera specific for different portions of huntingtin detect a ∼350 kDa protein in lymphoblast cells
(A) Detection of huntingtin. Identical Western blot panels of lymphoblastoid cell protein extracts were probed with preimmune (Pre) or immune (I) sera. The left-hand panel shows the results obtained with a 1:500 dilution of antipeptide serum HP1; the central panel, with 1:500 dilution of anti-peptide serum HP12; and the right-hand panel, a 1:10,000 dilution of anti-fusion protein HF1. The position of the 200-kD molecular weight standard (myosin) is shown. The identity of the ∼200-kD protein band detected by the preimmune and immune rabbit sera is not known but also serves as a convenient marker protein in human lymphoblast protein extracts. The position of the huntingtin band detected in both lanes of each panel by the immune sera is indicated and is estimated to be ∼350 kD. (B) Detection of huntingtin is abolished by preincubation with antigen. Identical Western blot panels of lymphoblast protein extract were probed with immune sera at the concentrations given in Panel A with (+) or without (−) preabsorption of each antiserum (HP1, HP12, and HF1) with 12 µg/ml of its corresponding immunogen. The positions of the 200-kD marker protein (myosin) and of the specifically competed ∼350-kD huntingtin protein are indicated. The right-hand panel was deliberately overexposed to visualize the ∼250-kD putative huntingtin breakdown product that is also competed by immunogen. This band is located between two nonspecific bands of similar intensity that are not competed. (C) HD huntingtin is distinguishable from normal huntingtin. Identical Western blot panels of lymphoblast protein extracts from HD heterozygotes (Lane 1) and HD homozygotes (Lane 2) were probed with a 1:500 dilution of HP1 (left-hand panel) and HP12 (central panel), and 1:10,000 dilution of HF1 (right-hand panel). The length of the polymorphic CAG stretch in the DNA of each individual was determined by PCR analysis and is shown above each lane as the estimated number of CAG repeat units, (CAG)n. The position of the 200-kD marker (myosin) is shown and the products of the normal and HD alleles, located close to each other at ∼350 kD, are denoted by a square bracket.