![Fig. 4. (A) Immunoprecipitation of gDWnt5a from transfected 293 cells. 293 cells were transiently transfected and metabolically labeled with [35S]methionine and [35S]cysteine 48 hours after transfection. Cell lysates and conditioned media were immunoprecipitated with MoAb 5B6 bound to protein A sepharose. The immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by fluorography. The arrow denotes the migration of gDWnt5a monomers. Note that the majority of WNT-5a is present in the cell lysate fraction. (B) Immunoprecipitation of gDWnt5aHis6 with the 5B6 MoAb depletes the stimulatory activity of WNT-5a CM towards flASK cells. (1) CM from 293 cells transfected with control plasmid (gDpRK5b), (2) control CM incubated with 5B6-CPG (CPG), (3) Wnt-5a CM (gDWnt5aHis6 ), and (4) Wnt-5a CM depleted with 5B6-CPG. The flASK cells were cultured in duplicate wells containing HSC media supplemented with 25 ng/mL KL for 7 days. The mean cell number was calculated from duplicate wells and repeated in two independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/89/10/10.1182_blood.v89.10.3624/3/bl_0047f4b.jpeg?Expires=1724246444&Signature=v~b9lBL9QkTOYLBJQnC58pwLaZ-Cxg58LV2j1115E8YFXukg2i9TbKYcM2LQxOBrzP8qZFuzO3OKAoULjFly7U4cmENaLXrBAdhZsLqIQ6-gC1d-a1S7otT0GX2x0r~BIQ6aPIlWHlaru48jkKPxyza~XtgSY6d04i3huE6turbgbvr7kC09wtDxcXBq83pNYQRSvwfpPgzKwwZHX61m9POQT9P6ZZHTzR4WN6brrex1uQdRg-IKh4INoYMUBTlN~ksACdwnIxPTRGs9EyNB9x59IfCvdjAbN~vy530fTZs6hG4Fzmo9omZW7JYu2mqce78wVZDyi9N-TelES3JqRw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
(A) Immunoprecipitation of gDWnt5a from transfected 293 cells. 293 cells were transiently transfected and metabolically labeled with [35S]methionine and [35S]cysteine 48 hours after transfection. Cell lysates and conditioned media were immunoprecipitated with MoAb 5B6 bound to protein A sepharose. The immunoprecipitates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by fluorography. The arrow denotes the migration of gDWnt5a monomers. Note that the majority of WNT-5a is present in the cell lysate fraction. (B) Immunoprecipitation of gDWnt5aHis6 with the 5B6 MoAb depletes the stimulatory activity of WNT-5a CM towards flASK cells. (1) CM from 293 cells transfected with control plasmid (gDpRK5b), (2) control CM incubated with 5B6-CPG (CPG), (3) Wnt-5a CM (gDWnt5aHis6 ), and (4) Wnt-5a CM depleted with 5B6-CPG. The flASK cells were cultured in duplicate wells containing HSC media supplemented with 25 ng/mL KL for 7 days. The mean cell number was calculated from duplicate wells and repeated in two independent experiments.
