Figure 3.

HFR1 and LAF1 interact in vitro and in vivo. (A) In vitro pull-down assay of full-length HFR1 with other proteins. (Panel a) Five-hundred nanograms of target proteins were pull downed with HFR1 protein (1 μg each) and detected by anti-MBP antibody. (Panel b) Purified target proteins used in pull-down assays were resolved on SDS-PAGE. Asterisks indicate the input proteins. (B) Definition of HFR1 interaction domain that binds to LAF1. (Panel a) Schematic diagrams of target proteins [MBP-HFR1, MBP-HFR1(N), and MBP-HFR1(C)]. (Panel b) In vitro pull-down assay to define the interaction domain of HFR1. (Panel c) Purified target proteins used in pull-down assays were resolved on SDS-PAGE. Asterisks indicate the input proteins. (C) Identification of noninteracting LAF1 mutant protein. (Panel a) The amino acid sequence of the LAF1 R3 domain (GenBank accession no. NM_118688) was aligned with those of PAP1/AtMYB75 (GenBank accession no. NM_104541), AtMyb5 (GenBank accession no. U26935), OsMyb2 (GenBank accession no. D88618), and ZmMyb8 (GenBank accession no. AM156905). Two amino acid residues, W87 and R97 (shaded), were mutated to Ala (A). The number of amino acids is indicated at the left and right sides. (Panel b) In vitro pull-down assays of LAF1(W87A) and LAF1(R97A). (Panel c) Purified target proteins used in pull-down assays were resolved on SDS-PAGE. (D) Coimmunoprecipitation of HFR1 with LAF1 protein. Protein extracts of double transgenic Arabidopsis seedlings [35S-HFR1-3HA and XVE-LAF1-6Myc or 35S-HFR1-3HA and XVE-LAF1(R97A)-6Myc] treated with MG132 (50 μM) plus β-estradiol (10 μM) were immunoprecipitated with anti-HA. Input proteins and the immunoprecipitates were separated on 10% SDS–polyacrylamide gels, blotted onto membranes, and detected with anti-HA and anti-Myc antibodies. Input refers to the starting protein amount in extracts used for immunoprecipitation reactions. The arrowhead indicates the cross-reaction with the heavy chain of the protein A-conjugated antibody.