Figure 4

Different 14-3-3 isoforms bind to the PRD1 region of POB1 in a phospho-dependent manner. A) Lysates of HEK293 cells, transiently transfected with POB1 fused to GFP, were incubated with four different isoforms of human 14-3-3 fused to GST adsorbed to glutathione-sepharose 4B beads. The retained proteins were separated by SDS-PAGE and probed with anti-GFP antibody (for POB1). 5% of the extract used in the pull down was loaded in the input lane. B) HEK293 cells were transiently transfected with different POB1 deletion constructs fused to GFP: POB1 full length (1–521 amino acids), POB1 EH domain (126–252), POB1 proline rich region 1 PRD1 (308–365), POB1 pro-rich and carboxy-terminal (308–521) and POB1 proline-rich region (308–398). Cell lysates were incubated with GST-14-3-3 ζ and GST alone, adsorbed to sepharose resin. The retained proteins were separated by 10% SDS-PAGE and probed with anti-GFP antibodies (for POB1). 5% of the extract adsorbed to the affinity resins was analyzed in the input lane. Figures in the deletion constructs refer to the amino acid residues of the POB1, short isoform, Swiss Prot entry Q8NFH8-2. The Ponceau staining shows the GST fusions and the input lysates. C). HEK293 cells were transiently transfected with vectors expressing GFP protein alone (panel above), POB1 PRD1 308–365 (middle panel) or full length POB1 (below) fused to GFP and treated as in B. 50% of each cell lysate was incubated with the Ser/Thr λ-phosphatase for 2 hours at 30°C before incubation with GST-14-3-3 ζ. Input lysates are 5% of the lysates adsorbed to the affinity resin. POB1 PRD1 and the full length POB1 are revealed by anti-GFP antibody. Upper panel is a negative control of mock vector expressing only GFP. D) A plasmid expressing Myc tagged POB1 was used to transfect HEK293 cells in two independent replica experiments. Cells extracts expressing the empty plasmid (lane 1) or the Myc tagged POB1 (lanes 2 and 3) were immunoprecipitated with anti-Myc antibodies (for POB1). The proteins immunoprecipitated by anti-Myc (20% of the total lysate) were separated by SDS PAGE and probed with anti-14-3-3 (upper panel) or anti-Myc antibodies (second panel). In the upper panel, the endogenous 14-3-3 proteins co-immunoprecipitating with POB1 are revealed by anti-14-3-3. Also the slower migrating band of the longest 14-3-3 isoform, epsilon, is efficiently recovered. The control of the immunoprecipitated POB1 is shown in the second panel. The endogenous 14-3-3 proteins in the input lysate are revealed by anti-14-3-3 in the third panel where the slower migrating band of the longest 14-3-3 isoform epsilon is also visible (5% of total lysate). The ectopically expressed POB1-Myc is revealed with anti-Myc antibodies, in the bottom panel (1% of total lysate).