Skip to main content
. 2017 Jan 31;3:17003. doi: 10.1038/celldisc.2017.3

Figure 1.

Figure 1

CARF is a target of NC043. (a) Chemical structures of NC043 and its analogs. (b) Effects of NC043 and its analogs on TOPFlash reporter activity. After 18 h of transfection, HEK293 cells were treated with the control (Ctr) conditioned medium (CM) or the Wnt3a CM plus the indicated small molecules for an additional 6 h before the luciferase activity assays. RLA, relative luciferase activity. Error bars indicate the s.d. of triplicate assays in one experiment. Each experiment was repeated at least three times. (*P-value<0.05; **P-value<0.01; ***P-value<0.001). (c) S-614 binds to CARF overexpressed in HEK293T cells. HEK293T overexpressed HA-tagged CARF were treated with 7.5 μm S-616 or S-614 plus with 0, 3.75 and 15 μm of NC043 for 1 h. The cell lysates were used for biotin-pulldown assay. The levels of CARF before and after pulldown were detected through western blotting analysis with HA antibody. (d) S-614 binds to endogenous CARF. After 1 h of 7.5 μm S-616 or S-614 plus with 0, 3.75 and 15 μm NC043 treatment, HEK293 cells were lysed, and followed by biotin-pulldown and western blotting analysis using a CARF specific antibody (anti-CARF). S-616 was used as a negative control. Error bars indicate the s.d. of triplicate assays in one experiment. Each experiment was repeated at least three times. (e) S-614 directly binds to purified CARF expressed in bacteria. The lysate of His6-tagged hCARF expressed E.coli. was incubated with Ni-beads for 1 h. After three times of wash, the beads were resuspended and incubated with small compounds (7.5 μm S-614 or S-616 and 15 μm NC043) as indicated for 1 h at 4 °C. Samples were subjected to biotin and His detaction. (f) NC043 binds to CARF covalently. After transfection, HEK293T cells were treated with 7.5 μm S-614 or S-616 and 15 μm NC043 as indicated, followed by biotin-pulldown and western blotting analysis. ‘*’ Cells were incubated in DMSO-contained medium for half-an-hour, and then treated with S-616/DMSO, S-614/DMSO or S-614/NC043 (lane 1-3) for 1 h; ‘**’ After S-614 pre-treatment, cells were incubated in S-614/DMSO or S-614/NC043 (lane 4–5) supplemented medium for additional 1 h. (g) Mapping the region on CARF responsible for binding NC043. Upper panel: schematic representation of full-length CARF (FL) and its truncations. After plasmids transfection and compounds incubation, HEK293T cells were used for biotin-pulldown and western blotting analysis. (h) C516 is the critical site for CARF to bind NC043. HEK293T cells expressed CARF (WT) and the indicated mutants were used for S614 binding assay followed by western blotting analysis.