Journal: Scientific Reports

Article Title: The most common RNF43 mutant G659Vfs*41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis

doi: 10.1038/s41598-019-54931-3

Figure Lengend Snippet: Expression, localization, RSPO binding, and stability of RNF43-G659Vfs*41 is similar to RNF43-WT. ( a ) Schematic diagram of domain structures of RNF43-WT and RNF43-G659Vfs*41. ECD, extracellular domain; TM, transmembrane domain; RING, RING E3 ligase domain. ( b ) WB of HEK293T cells transfected with vector control (Vec), or HA-tagged RNF43-WT, -G659x, or -G659Vfs*41 using anti-HA antibody. ( c ) Confocal immunofluorescence microscopy of STF-RZ-DKO cells transfected with RNF43-WT and -G659Vfs*41 and stained with mouse anti-HA and Alexa488-labeled anti-mouse IgG (green), or incubated with RSPO2-Fu-F109A-Fc followed by staining with Alexa-555-labeled anti-human IgG (red). Nuclei were counter-stained with TO-PRO-3 Iodide (blue). ( d ) Anti-HA WB of STK-RZ-DKO cells co-transfected with RNF43-WT and –G659Vfs*41 at 1:1 ratio and treated with cycloheximide for the indicated periods of time. ( e ) Quantification of RNF43 in WB of ( d ) normalized over GAPDH. ( f ) Anti-HA WB of STF-RZ-DKO, DLD, and LoVo cells transfected with increasing amounts of DNA of HA-tagged RNF43-WT and G659Vfs*41 plasmids pre-mixed at 1:1 ratio. GAPDH and actin were loading controls.

Article Snippet: Recombinant full-length human RSPO2 was purchased from R&D Systems.

Techniques: Expressing, Binding Assay, Transfection, Plasmid Preparation, Immunofluorescence, Microscopy, Staining, Labeling, Incubation

Journal: Scientific Reports

Article Title: The most common RNF43 mutant G659Vfs*41 is fully functional in inhibiting Wnt signaling and unlikely to play a role in tumorigenesis

doi: 10.1038/s41598-019-54931-3

Figure Lengend Snippet: RNF43-G659Vfs*41 inhibited Wnt signaling and reduced FZD levels as well as RNF43-WT could. ( a ) TOPFlash activity of STK-RZ-DKO cells transfected with vector (Vec), RNF43-WT, or –G659Vfs*41 in response to RPSO2 treatment. ( b ) WB of cells from ( a ) with anti-HA antibody. ( c ) WB of FZD5 in STK-RZ-DKO cells transfected with vector, RNF43-WT, or –G659Vfs*41 with anti-FLAG and anti-HA antibody. ( d ) WB of FZD5 of HEK293T cells transfected with vector, RNF43-WT, or G659Vfs*41 and treated with vehicle or RSPO2-Furin-Fc proteins (R2Fu) at 10 µg/ml. 1 = vehicle; 2 = RSPO2Fu-WT; 3 = RSPO2Fu-F109A. Actin and GAPDP were loading controls.

Article Snippet: Recombinant full-length human RSPO2 was purchased from R&D Systems.

Techniques: Activity Assay, Transfection, Plasmid Preparation