Fig. 4

NRIP2 regulates the activity of the Wnt pathway via RORβ. a Analysis of NRIP2 binding to RORβ. Lysates from NRIP2-overexpressing SGC cancer cells or control cells were subjected to co-IP with myc-tag antibody agarose beads, followed by western blotting with anti-RORβ antibodies. Normal rabbit IgG agarose beads were used as a negative control. b Endogenous NRIP2 interacts with RORβ. Lysates from P1 cells without treatment with protease inhibitor cocktails were subjected to co-immunoprecipitation with RORβ antibodies or mouse IgG (control), followed by western blotting with anti-NRIP2 antibodies. Normal rabbit IgG agarose beads were used as a negative control. c Wnt activity in RORβ-expressing cells. Wnt activity was assessed by Top/Fop flash reporter assays in SGC7901 cells 24 h after transient transfection with RORB or control plasmids. Overexpression of RORβ attenuated Wnt activity. *p < 0.05 (ANOVA). d Detection of Wnt dowmstream targets in RORβ-expressing cells. c-Myc and cyclin D1 were detected by western blotting in the above RORβ-overexpressing SGC7901 and control cells. e Detection of RORβ in RORB-knockdown cells. Cells were infected with lentivirus encoding RORB shRNA for 72 h and subsequently screened with 5 μg/mL Puromycin for 7 days. The surviving cell clone was picked out with limiting dilution analyses. RORβ was detected by western blotting in these RORB-knockdown clones, with cells infected with scrambled shRNA lentivirus as controls. f NRIP2 failed to activate the Wnt pathway in RORB-knockdown cells. Wnt activity was assessed by Top/Fop flash reporter assays in RORB-knockdown SGC7901 cell clones 24 h after transient transfection with NRIP2 or control plasmids. NRIP2 could not activate the Wnt pathway after knockdown of RORB. *p < 0.05 (ANOVA), ns: non-significance, p > 0.05 (ANOVA)