Figure 3.

Titration analysis of the effect of SMN self-association on SmB binding. (a) GST-SMN was preincubated with the indicated molar excess of His-tagged SMNwt, SMNY272C, or SMNΔEx7. After washing unbound recombinant proteins, in vitro translated [³⁵S]methionine-labeled SmB was added and binding was performed as described in Materials and Methods. Each binding was analyzed by Western blotting with anti-T7 tag mAb to show bound His-tagged SMN proteins (Upper) and by autoradiography to show bound SmB (Lower). (b) GST-SMNY272C was preincubated with the indicated molar excess of His-SMNY272C and processed further as described above. The input lane shows an amount equivalent to the 20% of SmB translation and to 1× His-SMNwt recombinant protein used.