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. 2009 Jan 21;28(2):135–143. doi: 10.1038/emboj.2008.271

Figure 6.

Figure 6

Constitutively active Ofd1 mutants are defective for binding to Nro1. (A) sre1N, sre1N ofd1-H210A, sre1N ofd1-H142A and sre1N ofd1-H142A D144A cells containing empty vector or nro1+ plasmid were treated with either 1% DMSO or 20 mM DMOG for 3 h. Whole-cell extracts (40 μg) were subjected to western blot analysis using anti-Sre1 and anti-Nro1 antibodies. (B) sre1N, sre1N ofd1-H210A, sre1N ofd1-H142A, sre1N ofd1-H142A D144A and sre1N ofd1Δ cells cultured in rich medium were treated with 2 mM DSP crosslinker in PBS for 5 min. Detergent-solubilized whole-cell extracts were subjected to immunoprecipitation with anti-Ofd1. Bound (10-fold overloaded) and unbound fractions were analysed by western blot with anti-Nro1–HRP and anti-Ofd1–HRP antibodies.