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Figure 1


Fig. 1. The N-terminus of the HRSL3 protein (HREV1) is important for the binding of HRSL3 and PR65. (A) Left panel: proteins were immunoprecipitated with an anti-V5 antibody, and subsequently detected with anti-HA (top) and anti-V5 (bottom) antibodies. Lanes (from the left): 1, total cell lysate; 2, immunoprecipitation from cellular extracts harboring both proteins; 3, 4, immunoprecipitation from cellular extracts harboring PR65{alpha}V5 or HREV1dCHA, respectively. Right panel: proteins were immunoprecipitated with an anti-HA antibody and subsequently detected with anti-V5 (top) antibody. The efficiency of the immunoprecipitation was controlled using an anti-HA antibody (bottom). Lanes (from the left): 1, immunoprecipitation from cellular extracts harboring both proteins; 2, 3, immunoprecipitation from cellular extracts harboring PR65{alpha}V5 or HREV1dCHA, respectively. (B) To prove whether PR65 directly interacts with HRSL3, cleared COS-7 cell lysates were incubated with either GST or GST-HREV1dC proteins coupled to GST-Sepharose. Relative amounts of the proteins were analyzed using western blotting against PR65 and GST antibodies. (C) The HREV1FL expression construct carries wild-type full-length HRASL3 cDNA. Four domains of the HRSL3 protein are indicated: a proline-rich domain at the N terminus of the protein (PD), a HWAIY domain containing a conserved histidine (His23), an NCE domain containing a conserved cystein (Cys112), and a membrane binding domain (MBD). In the HREV1dCHA expression construct, 29 C-terminal amino acids are substituted by the HA-epitope. AWAIdCHA carries a mutation His23->Ala; NSEdCHA carries a mutation Cys112->Ser; dNdCHA harbors a deletion of the N-terminal proline-rich region. (D) COS-7 cells were cotransfected with the following expression plasmids: PR65{alpha}V5 and HREV1dCHA, PR65{alpha}V5 and HREV1-AWAIYdCHA, PR65{alpha}V5 and HREV1-NSEdCHA, and PR65{alpha}V5 and HREV1-dNdCHA. As negative controls, plasmids containing HA or V5 epitopes only were transfected (–). (Left panel) Top: proteins were immunoprecipitated with an anti-V5 antibody and subsequently analyzed with an anti-HA antibody. HREV1dCHA, HREV1-AWAIdCHA and HREV1-NSEdCHA associate with PR65{alpha}V5, whereas the HREV1-dNdCHA deletion mutant is unable to interact with PR65{alpha}V5. Bottom: incubation with an anti-V5 antibody demonstrates equal amounts of the immunoprecipitated PR65{alpha}V5 protein. (Right panel) Top: proteins were immunoprecipitated with an anti-HA antibody and subsequently analyzed with an anti-V5 antibody. The PR65{alpha}V5 protein was obtained in a complex with HREV1dCHA, HREV1-AWAIdCHA and HREV1-NSEdCHA. By contrast, no PR65{alpha}V5 was detected in the immunoprecipitated HREV1-dNdC complex. Bottom: immunoprecipitation was controlled using an anti-HA antibody and demonstrates equal amounts of the HA-tagged HRSL3 proteins.





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