Figure 2

A) The SH3 domain of Amphiphysin II and the carboxy-terminal SH3 of Grb2 bind full length POB1 in pull down experiments. Lysates of 293 human embryonic kidney cells (HEK293), transiently transfected with POB1-Myc, were incubated with GST-SH3 of Amphiphysin II (AmphIISH3), GST-SH3 ammino-terminal of Grb2 (Grb2SH3-N), GST-SH3 carboxy-terminal of Grb2 (Grb2SH3-C) and GST alone, adsorbed to glutathione-Sepharose 4B beads. The retained proteins were separated by SDS-PAGE and probed with an anti-Myc antibody (for POB1). The input is 10% of the affinity-selected lysate. The AmphiphysinII SH3 binds 25% of the input while the carboxy-terminal SH3 of Grb2 binds 4%. The ammino-terminal SH3 of Grb2 does not bind POB1. The Ponceau staining shows the GST fusions and the input lysates. B) The SH3 interacting motif of POB1 is contained in the 308–365 region. HEK293 cells were transiently transfected with the indicated POB1 constructs expressed as fusions to GFP protein: GFP (lanes 1, 2, 3), GFP-POB1 full length (lanes 4, 5, 6) and GFP-PRD1, the poly-proline rich PRD1 region of POB1 from residue 308 to 365 (7, 8, 9). Cellular extracts were incubated with GST-SH3 of AmphiphysinII (left) or GST-SH3 carboxy-terminal of Grb2 (right) and GST alone. The retained proteins were subjected to SDS-PAGE and probed with an anti-GFP antibody (for POB1 or PRD1). The sample loaded in the input lane is 5% of the material used for affinity selection. A faint unspecific band is visible between the POB1 full length and the PRD1. POB1 PRD1 (308–365) is bound by both the SH3 tested: Amphiphysin II (last lane in leftmost gel) and Grb2 (last lane in rightmost gel). The Ponceau staining shows the GST fusions and the input lysates. C) POB1 full length co-immunoprecipitates with full length Amphiphysin II and endogenous Grb2. Left: POB1-GFP was co-expressed in HEK293 cells together with Grb2-HA or Amphiphysin II-FLAG (Amph-FLAG). Cellular extracts were immunoprecipitated (IP) with anti-HA antibody (for Grb2) or anti-FLAG resin (for Amphiphysin II), respectively and the co-immunoprecipitated POB1 was revealed with an anti-GFP antibody. Two lanes for each immuno-precipitation correspond to the same experiment repeated twice but at different washing stringency (without or with 1% Triton). Mock HA and FLAG lanes represent controls transfected with empty vectors. In the input lane we have loaded 1.5% of the cell lysate used for the immunoprecipitation. Right: An empty vector (lane 1) and two independent transfections of POB1-Myc were carried out in HEK293 cells (lanes 2, 3). Upper panel: POB1-Myc in cell lysate was revealed with an anti-Myc antibody (5% of the total cell lysate). Second panel: Cells extracts were immunoprecipitated with anti-Myc (for POB1) and the immunoprecipitated POB1 (an amount corresponding to 20% of the total lysate) were revealed with an anti-Myc antibody; Third panel: the cell lysate was probed with anti-Grb2 antibody, to detect endogenous Grb2 (1% of the total lysate). Panel below: Cells extracts were immunoprecipitated (IP) with anti-Myc (for POB1) and the co-precipitated material was blotted (WB) with an anti-Grb2 antibody to detect endogenous Grb2 co-immunoprecipitated by POB1.