Ab150358

Standard western blot techniques were performed as previously described (27 (link)) using NuPAGE 4–12% Bis-Tris Protein Minigels (Invitrogen). The antibodies used are listed below. Primary antibodies: 1) Podocalyxin-like 1 (3D3) mouse monoclonal antibody (Santa Cruz Biotechnology, sc-23904, 1:500). 2) PODXL (EPR9518) rabbit monoclonal antibody (Abcam, ab150358, 1:1000). 3) Dynamin-2 (DYN2–11) mouse monoclonal antibody (Sigma Aldrich, SAB4200661, 1:500). 4) Dynamin-2 rabbit polyclonal antibody (Abcam, ab3457, 1:1000). 5) Ezrin (3C12) mouse monoclonal antibody (Abcam, ab4069, 1:500). 6) NHERF2 (D3A5) rabbit monoclonal antibody (Cell Signaling, 9568, 1:1000). 7) GST (26H1) mouse monoclonal antibody (Cell Signaling, 2624, 1:2000) 8) 6x-His tag (4E3D10H2/E3) mouse monoclonal antibody (ThermoFisher Scientific, MA1–135, 1:2000). 9) RhoA (7F1.E5) mouse monoclonal antibody (Cytoskeleton, ARH04, 1:500). 10) Rac1 mouse monoclonal antibody (Cytoskeleton, ARC03, 1:500). 11) Actin (C4) mouse monoclonal antibody (BD Transduction, 612656, 1:10000). Secondary antibodies: 1) Anti-mouse IgG, HRP-linked antibody (Cell Signaling, 7076S, 1:2000). 2) Anti-rabbit IgG, HRP-linked antibody (Cell Signaling, 7074S, 1:2000).

Co-IP was performed to identify the binding partner(s) of PODXL in pancreatic cancer cells with Pierce co-IP Kit (Thermo Scientific). The co-IP assays were performed by incubating pre-cleared cell lysates containing 500 μg of total protein with 25 μl of resin immobilized with 10 μg of antibody overnight at 4°C. PODXL (EPR9518) rabbit monoclonal antibody (Abcam, ab150358) was used to immunoprecipitate PODXL. A rabbit IgG monoclonal isotype (EPR25A, Abcam, ab172730) or beads-only controls were included to account for non-specific binding. Unbound proteins were washed away from the antibody-immobilized resin by centrifugation with IP lysis buffer supplemented with 1M NaCl for 10 times. Bound proteins were eluted with 1% SDS and concentrated. The co-IP elution was submitted to the Johns Hopkins Mass Spectrometry and Proteomics Core Facility (Baltimore, MD, USA) for protein identification or analyzed with standard western blot protocol once the candidate protein was identified. In subsequent reciprocal co-IP experiments, Dynamin-2 rabbit polyclonal antibody (Abcam, ab3457) or NHERF2 (D3A5) rabbit monoclonal antibody (Cell Signaling, 9568) were used to immunoprecipitate dynamin-2 or NHERF2, respectively.