gfp trap  (Proteintech)

Figure Legend Snippet: DKC1125 inhibits the function of Dvl by interfering with the interaction of KSRP with Dvl2. a Association of KSRP with Dvl2 was inhibited after treatment of DKC1125. Co-IP experiments were performed in Caco2 cells stably expressing EV or KITENIN-V5 using cytoplasmic–nuclear fractionation after treatment with vehicle (V) or DKC1125 treatment (0.5 μM). Precipitates were analyzed by immunoblotting to detect KSRP-Dvl binding. Whole-cell lysate (WCL) of the same pool of cells was co-analyzed as a control. b The amount of Dvl2 protein adhering to KSRP was markedly reduced by treatment with DKC1125. Dvl2-ΔDIX-GFP transfected HCT116 cells were collected and lysed, and the supernatants were mixed with purified KSRP-His by Ni-NTA after in vitro bacterial expression and subjected to GFP-Trap. GFP-Trap, consisting of an anti-GFP Nanobody/VHH coupled to agarose beads, was used for effective pulldown of GFP-fusion proteins. Interaction of ΔDIX-DVL2-GFP with bacterially expressed KSRP-His was verified by immunoblotting with anti-His antibody. c Effect of DKC1125 on Dvl binding of WT-KSRP or ΔKH34-KSRP. Co-IP analysis was performed using cytoplasmic–nuclear fractions obtained from WT-KSRP or ΔKH34-KSRP transfected Caco2 cells stably expressing KITENIN-V5 or EV. Binding was verified by immunoblotting. d Effect of DKC1125 on activation of WNT/β-catenin by expression of KITENIN and/or KSRP. 293 T cells were transfected with the TOP-flash reporter gene and KITENIN-V5, WT-KSRP-myc, or ΔKH34-KSRP-myc, in parallel, and treated with vehicle or DKC1125 (0.5 μM). Luciferase activity was measured at 48 h after transfection and normalized against the activity of TK-Renilla. Data are shown relative to the corresponding TOP-flash value in control cells. Differences in transcriptional activity of TCF/LEF by expression of KITENIN, WT-KSRP, and ΔKH34-KSRP were compared between the presence and absence of DKC1125. Error bars indicate SEM. The asterisk indicates a significant difference between groups (NS, not significant; ** P < 0.01)

Techniques Used: Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Fractionation, Western Blot, Binding Assay, Control, Transfection, Purification, In Vitro, Activation Assay, Luciferase, Activity Assay

Figure Legend Snippet: Treatment with DKC1125 results in autophagic degradation of Dvl2 and KITENIN through increased binding to RACK1. a RACK1 directly binds to DKC1125. Proteins pulled down by chemical probe using HCT116 lysates were verified by immunoblot analysis using antibodies against RACK1 and Dvl2. b Interactions of RACK1 with KSRP or Dvl2 within the functional KITENIN complex following DKC1125. Caco2 cells were transfected with empty vector (EV) or KITENIN-V5 (KIT-V5), and treated with vehicle (V) or DKC1125 (DKC) (0.5 μM). The cell lysates were immunoprecipitated with anti- RACK1 antibody and immunoblotted with anti-KSRP or anti-Dvl2 antibody. c Levels of Dvl2 and KITENIN are reduced more after DKC1125 treatment under RACK1 expression. Caco2 cells were transfected with empty vector (EV) or KITENIN-V5, or co-transfected with KITENIN-V5 and RACK1-GFP, and then treated with vehicle or DKC1125 (0.5 μM). The protein levels of Dvl2 and KITENIN were checked after treatment with cycloheximide at the indicated times. d RACK1 affects the activation of canonical WNT signaling by Dvl2. 293 T cells were transfected with the TOP-flash reporter gene and Dvl2, RACK1, or si-RACK1, in parallel, and treated with vehicle or DKC1125 (0.5 μM). Differences in transcriptional activity of TCF/LEF by Dvl2, alone or in combination with RACK1 overexpression or knockdown, were compared between the presence or absence of DKC1125. e Autophagic degradation of Dvl2 and KITENIN by DKC1125. Caco2 cells were initially pretreated with vehicle, the proteasome inhibitor MG132 (MG, 10 μM), the lysosomal degradation inhibitors bafilomycin A1 (A1, 100 nM) and chloroquine (CQ, 10 μM), or the autophagosome blocker type III phosphatidylinositol 3-kinase inhibitor (3-MA, 1 mM), and later treated with a high concentration of DKC1125 (5 μM). Levels of Dvl2 and KITENIN were examined by immunoblot analyses. f Staining of autophagosomes in DKC1125-treated cells. Autophagosomes were stained in stably KITENIN-expressing Caco2 cells after 24 h treatment with DKC1125 using CYTO-ID autophagy detection dye. Rapamycin was included as a positive control for autophagic induction

Techniques Used: Binding Assay, Western Blot, Functional Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Expressing, Activation Assay, Activity Assay, Over Expression, Knockdown, Concentration Assay, Staining, Stable Transfection, Positive Control

Figure Legend Snippet: RACK1 and miR-124 are required for the suppressive effects of DKC1125 on cell invasiveness. a RACK1 in the KITENIN complex plays a major role in the inhibition of cell invasion by DKC1125. Cell invasion was examined in empty vector (EV)- or KITENIN-transfected Caco2 cells after knockdown of RACK1 via siRNA transfection (left panel), or under ectopic expression of RACK1 (right panel) after treatment with vehicle (V) or DKC1125 (D) (0.5 μM). Data are expressed as in Fig. a . b Dvl2 downregulation by DKC1125 was associated with elevated binding of Dvl2 to RACK1. Several deletion mutants within the binding site of KSRP to DKC1125 were designed and co-expressed in Caco2 cells with RACK1-GFP. Dvl2–RACK1 binding was examined using GFP-Trap and immunoblot analysis after treatment with DKC1125 (0.5 μM), and compared with that of empty vector (EV) or wild-type (WT) KSRP expression. An increase in Dvl2–RACK1 interaction was observed after DKC1125 treatment in cells expressing the Q417A-KSRP or N467A-KSRP mutant, but not in cells expressing the R411A-KSRP or R415A-KSRP mutant. c Modulation of miR-124 is also involved in increased cellular invasiveness by the functional KITENIN complex. Detection of transcript of miR-124-3p in stably miR-null- or miR-124-transfected Caco2 cells (left panel). Cell invasion was examined in Caco2 cells stably expressing the miR-null vector or miR-124 that were transfected with the empty vector (EV)-, KITENIN-, WT-KSRP-, or Δ34KH-KSRP, and treated with vehicle or DKC1125 (0.5 μM) (right panel). Data are expressed as in Fig. a . d Inhibitor of miR-124-3p significantly restored the inhibitory effect of DKC1125 on the KITENIN-mediated increase in cell invasion. Cell invasion was compared in empty vector (EV)- or KITENIN-transfected Caco2 cells treated with vehicle or DKC1125, or co-treated with DKC1125 (0.5 μM) and a synthetic-oligo inhibitor of miR-124-3p (50 nM). The asterisk indicates a significant difference in Caco2/KITENIN-V5 cells after treatment with DKC1125, and a significant difference in DKC1125-treated Caco2/KITENIN-V5 cells after treatment with a synthetic-oligo inhibitor of miR-124-3p (** P < 0.01). Data are expressed as in Fig. a

Techniques Used: Inhibition, Plasmid Preparation, Transfection, Knockdown, Expressing, Binding Assay, Western Blot, Mutagenesis, Functional Assay, Stable Transfection