Figure 7. BLACAT2 directly binds to WDR5 protein and regulates VEGF-C expression.

(A) Representative image of silver-stained PAGE gels showing separated proteins that were pulled down using biotin-labeled BLACAT2. In vitro–transcribed antisense sequence of BLACAT2 was used as the nonspecific control. (B) Western blot analysis indicating that BLACAT2 associates with WDR5, as indicated by the pull-down assay with nuclear extracts or in vitro–synthesized WDR5. Antisense BLACAT2 was used as the negative control RNA in the pull-down assay. (C) RT-qPCR analysis of RNA enrichment in the RIP assay using the anti-WDR5 antibody in UM-UC-3 and 5637 bladder cancer cells. Normal IgG was used as the nonspecific control antibody. U1 and HOTTIP were used as negative and positive controls, respectively, for WDR5 binding. (D and E) Serial deletions of BLACAT2 were used in RNA pull-down assays to identify core regions of BLACAT2 that were required for physical interaction with WDR5. (F–I) Site-directed mutagenesis of 100–130 nt of BLACAT2 was performed, and the effects of BLACAT2 mutant overexpression on VEGF-C mRNA expression (F), VEGF-C secretion (G), HLEC migration (H), and HLEC tube formation (I) were evaluated. All experiments were performed with at least 3 biological replicates. Statistical significance was assessed using 1-way ANOVA followed by Dunnett’s tests for multiple comparisons (C, F–I). **P < 0.01.