Consistent with these data, PKC family inhibitor G?6983 had no effect on RIPK4-induced -catenin build up (data not shown). transfected with bare vector, wild-type RIPK4, or K51R mutant RIPK4 for 48 h. Error bars symbolize the s.e.m. of triplicate measurements. Results are representative of 3 self-employed experiments.B. Western blotting of HEK293T cells transfected with indicated plasmids for 48 h. NIHMS591305-supplement-Figure_S3.pdf (81K) GUID:?B0FDEBEE-5638-4200-96B5-96D5B5862526 Figure S4: Supplemental Figure 4. RIPK4 stimulates Wnt pathway activation individually of PKC A. HEK293T cells transfected with RIPK4-FLAG (wild-type or mutant K51R) were immunoprecipitated with anti-FLAG beads and immunoblotted for PKC- or PKC-. Non-specific bands (*).B. HEK293T cells were transfected with PKC siRNAs and RIPK4 plasmid as indicated for 48 h. NIHMS591305-supplement-Figure_S4.pdf (110K) GUID:?9F25947B-BBCA-471D-8A6F-61F1BF2A328A Number S5: Supplemental Number 5. Knockdown of RIPK4 compromises Wnt pathway in HEK293T cells A. HEK293T cells infected by RIPK4 or control shRNA were further transfected with the control or RIPK4 siRNAs and then treated with 150 g/ml Wnt3a.B. Cells from A were transfected with TOP-Brite luciferase reporter for 48 h and treated with 150 g/ml Wnt3a for 3h. CCD. Cells from A were treated with 150 g/ml Wnt3a for 10h. Transcripts levels of AXIN2 and APCDD1 were measured by QPCR. NIHMS591305-supplement-Figure_S5.pdf (106K) GUID:?6A68B875-0878-4484-A768-5CD74CBEE33E Number S6: Supplemental Number 6. DS18561882 morpholino phenotypes As with Fig. 2B, RNA was prepared from whole embryos or animal caps prepared from embryos injected animally DS18561882 in both blastomeres in the two-cell stage with either only or together with and control genes. NIHMS591305-supplement-Figure_S6.pdf (53K) GUID:?7C622AF4-FD61-43FE-AB54-8A1FD3805D96 Number S7: Supplemental Number 7. DVLs and LRP6 are required for RIPK4 to cause cytosolic -catenin build up A. HEK293T cells were transfected with the siRNAs indicated and RIPK4 plasmid for 48 h.B. TOPbrite luciferase activity in HEK293 cells transfected with the siRNAs indicated and RIPK4 plasmid for 48 h. Error bars symbolize the s.e.m. of triplicate measurements. Results are representative of 3 self-employed experiments. C. Control IP for Fig. 3A. NIHMS591305-supplement-Figure_S7.pdf (122K) GUID:?E98C1139-8977-46BF-A31A-40F8084C7C42 Number S8: Supplemental Number 8. RIPK4 phosphorylates DVL2 at Ser298 and DS18561882 Ser480 A. HEK293T cells were transfected with DVL2-FLAG and RIPK4-FLAG (wild-type or mutant K51R) for 48 h. Cells expressing wild-type RIPK4 were cultured in 13C6-lysine, 15N2-arginine weighty medium. Pooled lysates were immunoprecipitated with anti-FLAG beads and two DVL2 peptides phosphorylated only by wild-type RIPK4 were recognized by mass spectrometry.B. DVL proteins aligned around DVL2 phosphorylated residues Ser298 and Ser480. C. HEK293T cells transfected with bare vector or DVL2 (crazy type or mutants Ser298A, Ser480A, and Ser298A Ser480A) for 48 h. Whole cell lysates were western blotted with phospho-specific DVL2 antibodies. Non-specific bands (*). D. HEK293T cells transfected DNAJC15 with DVL2-FLAG and RIPK4-Myc (wild-type or mutant K51R) for 48 h. Where indicated, whole cell lysates were treated with calf intestinal alkaline phosphatase (CIP). E. HEK293T cells stably expressing control or shRNA were treated with 200 ng/mL Wnt3a. nonspecific bands (*). NIHMS591305-supplement-Figure_S8.pdf (121K) GUID:?ADF363A7-87D7-4517-9AD1-CD770C734302 Number S9: Supplemental Number 9. RIPK4 causes DVL2 to form puncta in HeLa cells Immunofluorescence microscopy of HeLa cells transfected with RIPK4-GFP and DVL2-FLAG for 36 h. Level bars: 10 m. NIHMS591305-supplement-Figure_S9.pdf (52K) GUID:?415775BC-1ED4-4C60-9F05-D0D6A8BDC44B Table S1. NIHMS591305-supplement-Table_S1.pdf (22K) GUID:?7500ECDD-2A0A-4D47-8CDA-4A1473C59A09 Abstract Receptor interacting protein kinase 4 (RIPK4) is required for epidermal differentiation (1C4) and is mutated in Bartsocas-Papas syndrome (5, 6). While RIPK4 binds protein kinase C (5, 6), RIPK4 signaling mechanisms are mainly unfamiliar. We display that ectopic RIPK4 induces cytosolic -catenin build up and a transcriptional system much like Wnt3a, whereas kinase-defective or Bartsocas-Papas syndrome RIPK4 mutants do not. Ectopic synergized with Wnt family member in morpholinos or kinase-defective RIPK4 antagonized Wnt signaling. Mechanistically, RIKP4 interacted constitutively with the Wnt adaptor protein DVL2 and, after Wnt3a activation, with the co-receptor LRP6. Phosphorylation of DVL2 at Ser298 and Ser480 by RIPK4 favored canonical Wnt signaling. Growth of a Wnt-dependent N-Tera2 xenograft tumor model was suppressed by knockdown, suggesting that overexpression may contribute to the growth of particular tumor types. in autosomal recessive Bartsocas-Papas syndrome also causes severe problems in face, pores and skin, and limb development (5, 6). To better determine the RIPK4 signaling pathway, we used a Human Transmission Transduction PathwayFinder PCR Array to determine gene manifestation changes in HEK293T cells after transfection with RIPK4. Canonical Wnt target genes such as were upregulated (fig. S1), prompting us to compare the effect of RIPK4 to Wnt3a treatment in.