![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=1735839382&Signature=DrYP7cZGG9AYnUdWu2TrZJ7ppfzNm0XbavPTm9blCy-K3OkHHzJN7u8dyPiF7YqX4brZuBPmXS7KXHCcT9CD~yrqAZyslk7-G853KPKOp05oikbSDKFfw8h1rtPNBU1u-a4FRdalZaGBKOzXNAJ2CltrhqNZX1eRN7TXFeusYjVMpKbLvHTSLxt3JZw4-S~2OZP9Xn0NnoliBGCCPhV-04Oe3AsfBHIKfu-K8GpxvLX3MTp3lD6pc1shmYZ7mzdOC-fBvz99Jvq0~N82nNYspfivIeBpax0Tt1QRjyIebRWk58735SpKz3C5Zc27mHo5hRwNAEjYwc1AkiOiyB36AA__&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.
