Fig 2: Electrophoretic migration of protein samples after three chromatographic steps. A, B, and C. 2 µl of concentrated samples (at 10 mg/ml) of PBX11–430:PREP11–436, PBX11–308:PREP11–344 and PBX11–317:PREP11–344 were loaded onto SDS PAGEs. The full-length proteins are shown in panel A and some degradation products are present. The C-terminal deletion mutants of high purity are shown on the panels B and C. D and E. Immunoblots of purified PBX11–430:PREP11–436 complex, where the N-terminal degradation of both PBX1 and PREP1 is evident.

Fig 3: DNA binding of purified recombinant PBX1:PREP1 complexes. A and B. EMSA of PBX1:PREP1 complex binding to O1 oligonucleotides. Co-purified full-length PBX1:PREP1 complex (2.5 µM) was incubated in the presence of 1 µg of poly(dIdC) at 1:1 molar ratio with a 22 bp DNA fragment (Panel A) or an 11 bp DNA fragment (Panel B) for 30 minutes at 4°C before loading on gel (Lanes 2). The sequences of DNA are reported in Table 2. As a reference the complex without DNA was also loaded on gel (Lanes 1). Note that with the 22 bp oligo (Panel B) at the highest ratios an additional upper band is visible, possibly corresponding to the binding of a second PREP1:PREP1 complex to the DNA. C and D. EMSA of PBX11–308:PREP11–344:DNA complex. Co-purified PBX11–308:PREP11–344 complex (2.5 µM) was incubated in the presence of 1 µg of poly(dIdC) at 1:1 molar ratio with the 22 bp (Panel C) or the 11 bp DNA fragment (Panel D) for 30 minutes at 4°C before loading on gels (Lanes 2). In Lanes 1 were loaded the protein complexes without DNA. E. Static light scattering analysis of the PBX11–308:PREP11–344 complex. The chromatograms show the UV absorbance in blue (scale on the left) and the calculated molecular mass in red (scale on the right). PBX11–308:PREP11–344 complex was analyzed in the absence (top) and in the presence (bottom) of the 11 bp DNA oligonucleotide

Fig 4: Expression and purification of recombinant PREP1 and PBX1. A. Expression and purification of full-length PREP1. SDS PAGE analysis of proteins at different purification stages. Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, pellet; Lane 4, cleared lysate; Lane 5,empty; Lane 6, first wash after incubation with glutathione beads (Tris-buffer with 0.5 M NaCl); Lane 7, second wash (Tris-buffer with 0.3 M NaCl); Lane 8, third wash (Tris-buffer with 0.3 M NaCl); Lane 9, glutathione beads before elution; Lane 10; glutathione beads after elution; Lane 11, supernatant from glutathione beads, containing the eluted protein. B. Expression and purification of full-length PBX1. Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, cleared lysate; Lane 4, flow-through after incubation with glutathione beads; Lane 5, first wash (Tris-buffer with 0.5 M NaCl); Lane 6, second wash (Tris-buffer with 0.3 M NaCl); Lane 7, glutathione beads before elution; Lane 8; glutathione beads after elution; Lane 9, empty; Lane 10, supernatant from glutathione beads, containing the eluted protein. C. Expression and purification of PREP1 1–344. Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, cleared lysate; Lane 4, flow-through after incubation with glutathione beads; Lane 5, first wash (Tris-buffer with 0.5 M NaCl); Lane 6, second wash (Tris-buffer with 0.3 M NaCl); Lane 7, third wash (Tris-buffer with 0.3 M NaCl); Lane 8, empty; Lane 9; glutathione beads before elution; Lane 10, glutathione beads after elution; Lane 11, empty; Lane 12, supernatant from glutathione beads, containing the eluted protein. D. Expression and purification of PBX1 1–317. Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, pellet; Lane 4, cleared lysate; Lane 5, flow-through after incubation with glutathione beads; Lane 6, first wash (Tris-buffer with 0.5 M NaCl); Lane 7, second wash (Tris-buffer with 0.3 M NaCl); lane 8, third wash (Tris-buffer with 0.3 M NaCl); Lane 9, glutathione beads before elution; Lane 10, glutathione beads after elution; Lane 11, empty; Lane 12, supernatant from glutathione beads, containing the eluted protein. E. Co-expression and purification of GST-PBX1 1–430 PREP1 1–436. Lane M, Bio-Rad size standard; Lane 1, total lysate before IPTG induction; Lane 2, total lysate after IPTG induction; Lane 3, pellet; Lane 4, cleared lysate; Lane 5, flow-through after incubation with glutathione beads; Lane 6, first wash (Tris-buffer with 0.5 M NaCl); Lane 7, second wash (Tris-buffer with 0.3 M NaCl); Lane 8, third wash (Tris-buffer with 0.3 M NaCl); Lane 9, glutathione beads before elution; Lane 10, glutathione beads after elution; Lane 11, supernatant containing the eluted proteins.

Fig 5: Confocal analysis of PREP1-GFP HR1m and HR2m subcellular localization. Panel A: Schematic representation of PREP1-GFP constructs used for immunofluorescence and pull-down experiments. In the scheme, the deleted regions are indicated along with the positions of the quadruple PREP1 mutations HR1m (L63A/L66A/L67A/L70A) and HR2m (I122A/L125A/L129A/L132A). Panel B: PBX1 pull-down in total lysates of A549 cells overexpressing wild-type or mutants PREP1-GFP. This panel shows cropped parts of a single immunoblot membrane (the full-length blots are shown in Supplementary Information Figure S4D) stained with different antibodies. The top part shows the anti-vinculin staining, the middle part the anti-GFP and the bottom part the anti-PBX1 staining, as indicated. In all cases, lanes 1–6 are the pull-down inputs; lanes 7–12 the pull-down results. All lysates were supplemented with a total of 1 µg of purified recombinant PBX1. Lanes 1 and 7: lysates of PREP1-GFP wild-type expressing cells; lanes 2 and 8, lysates of PREP1-GFP L63A/L66A/L67A/L70A expressing cells (HR1m); lanes 3 and 9 lysates of PREP1-GFP I122A/L125A/L129A/L132A (HR2m) expressing cells; lanes 4 and 10 lysates of PREP1-GFP ?HR1 + 2 expressing cells; lanes 5 and 11 lysates of cells expressing GFP alone; lanes 6 and 12 lysates of PREP1-GFP ?HD expressing cells. Panel C: A549 cells were transfected with a plasmid encoding wild-type, or mutants (HR1m, HR2m, ?HR1 + HR2, or ?HD) PREP1-GFP. The figure shows illustrative confocal immunofluorescence microscopy images in which the nucleus is identified by DAPI fluorescence and PREP1 by GFP immunofluorescence. The transfected constructs are indicated on the left. PREP1-GFP is in green, DAPI in blue, phalloidin in red. The merged DAPI, GFP and red channels are shown on the right. PREP1-GFP mutants reveal a cytoplasmic localization, compared to the exclusively nuclear localization of PREP1-GFP wild-type. Panel D: Bar graphs showing the intracellular distribution of wild-type and mutants PREP1-GFP. Plotted is the normalized nuclear/cytoplasmic GFP quantification, using a minimum 32 individual cells for each construct.The number of cells quantified is reported at the bottom of each histogram bar. T-TEST was performed as described in the Methods section.