Fig. 1.
Western blots of HeLa cell lysates expressing ataxin-1 (Q2, 30 or 82)-GFP. (A and B) Time course crosslinking of ataxin -1-GFP by TG2. Immunoreactive bands were detected with anti-polyglutamine (A) and anti-GFP (B) antibodies. When indicated, 10 μl aliquots were removed and electrophoresed on a reducing 4–20% polyacrylamide gel. In controls (A) TG2 was omitted except ‘Enz’ (enzyme) lane. The reaction produced large multimers, which stayed on top of the gel. (C) Silver stained gel after 0 and 30 min incubations with TG2. (D and E) GFP immunoblots showing inhibition of ataxin-1 aggregate formation in the presence of various concentrations of TG2 inhibitor cystamine. In ‘E’ the reaction mixtures were incubated for 10 min with guinea pig TG2. Lysate prepared from HeLa cells transfected with GFP without extra Q tract (D) shows no crosslinking.