|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 3. Confirmation of the interaction of PSGL-1 with versican. (A) G3, CRDCBP and CBP were incubated with glutathione S-transferase (GST), GST fused with PSGL-1 or fused with amino acids 279-402 of PSGL, GST(279-402). The mixture was purified using GST affinity columns, and purified products were analyzed on a western blot probed with 4B6 that recognizes an epitope engineered in each construct. Only G3 product interacted with both fusion proteins, GST-PSGL and GST(279-402). (B) HL60 cells that express endogenous PSGL-1 were incubated with medium containing G3, CRDCBP or CBP. HL60 cell lysate prepared by sonication was incubated with protein G beads pre-incubated with bovine serum to avoid non-specific binding or protein G saturated with 4B6. After washing, proteins binding to the beads were subjected to western blotting with anti-PSGL-1 antibody. Untreated lysate served as a control. Only G3 interacted with endogenous PSGL-1. (C) Human leukocytes were lysed and centrifuged. The supernatant was subjected to immunoprecipitation using 2B1. After washing, the bound proteins were analyzed on a western blot probed with anti-PSGL-1 antibody to detect the interaction of endogenous versican and PSGL-1.
|
