Fig. 3. PODXL interacts with dynamin2 in pancreatic cancer cells.

(A) Ezrin was not detected in the immunoprecipitate fraction generated from SW1990 pancreatic cancer cells using a PODXL specific antibody. An IgG isotype antibody was used as a control. The right lane shows the presence of both PODXL and ezrin in SW1990 cell lysate input. (B) Co-IP experiments from SW1990 cell lysates were performed using either a PODXL (left panels) or a dynamin-2 (right panels) specific antibody along with matched isotype controls. The resultant immunoprecipitates were subsequently resolved with SDS-PAGE and blotted with antibodies against PODXL and dynamin-2. Dynamin-2 was detected in the immunoprecipitates of PODXL and vice versa. The right lanes in both panels reveal the presence of both PODXL and dynamin-2 in SW1990 cell lysate inputs. The left lanes in both panels show the beads-only (no antibody) controls. (C) His-tagged constructs encompassing different domains of dynamin-2 were used for the in vitro His-tag pulldown assay. Mid, middle domain; PH, pleckstrin homology domain; GED, GTPase effector domain; PRD, proline-rich domain. (C) In-vitro His-tag pulldown assays demonstrating that the GTPase, middle and pleckstrin homology domains of dynamin-2 are responsible for binding PODXL from SW1990 cell lysates. * denotes the respective His constructs. (E) In-vitro His-tag pulldown assay showing that His-tagged PODXL cytoplasmic tail (PCT) is sufficient to bind to endogenous dynamin-2 from SW1990 cell lysates. (F) In-vitro His-tag pulldown assay demonstrating that purified GST-tagged PCT binds to recombinant His-tagged dynamin-2 D3+D4 domains but not to D4 alone (left panel). Purified GST alone does not bind to either His-tagged constructs. # denotes the protein band of GST (28kDa) from previous immunoblotting with anti-GST antibody.