Figure 1

Association of uPAR with HSP70 and regulation of the uPAR/HSP70 complex by MRJ. (A) Co-IP of uPAR and HSP70 in HCT116 cells. Cell lysates were immunoprecipitated with anti-uPAR antibody and IgG and the Western blot was probed with anti-HSP70 antibody. (B) Co-IP of uPAR and HSP70 in HEK293 cells co-transfected with HSP70-flag and uPAR-HA constructs or transfected with uPAR-HA (top lanes) or HSP70-flag alone (down lanes). IP was performed using anti-HA or anti-flag antibody and WB was performed using anti-flag antibody (upper blot) or anti-HA antibody (lower blot). 6% or 2% of the amount of the original cell lysates used for IP was loaded as an input control and visualized with anti-HA antibody (top lanes) or anti-flag antibody (down lanes), respectively. (C) Over-expression of MRJ increased HSP70 in HEK293 cells. The cells were transfected with uPAR-HA, or uPAR-HA plus HSP70-flag or uPAR-HA plus HSP70-flag plus MRJ-flag. IP was performed using anti-HA antibody and WB was then performed using anti-flag to detect the indicated proteins. Equal amounts of protein in each cell lysate were blotted with anti-HA antibody as controls. (D) Knockdown of MRJ decreased the HSP70 in HEK293T cells. The cells were co-transfected with uPAR-HA and HSP70-flag, with or without psiMRJ. Co-IP was performed using anti-HA antibody and WB was performed using anti-flag to detect the indicated proteins. Equal amounts of protein in each cell lysate were blotted with anti-HA antibody as controls. (E) HSP70 regulated the interaction among HSP70 and MRJ and uPAR. The cells were co-transfected with uPAR-HA plus MRJ-flag, or uPAR-HA plus MRJ-flag plus HSP70-prk5, or uPAR-HA plus MRJ-flag plus psiHSP70. Co-IP was performed using anti-HA antibody and WB was performed using anti-flag to detect the indicated proteins. Equal amounts of protein in each cell lysate were blotted with anti-HA antibody as controls.