Fig. 5

Identification of HuD protein and GRB-10 / ARL6IP1 RNA interacting domains in neuroblastoma cells. A GRB-10 and ARL6IP1 mRNA capture by recombinant GST-HuD; assay via RT-qPCR. B Biotinylated RNA oligoes specific to GRB-10 and ARL6IP1 were used to pulldown HuD protein by Western blot in IMR-32 and SK-N-SH cell lines. Nonspecific oligo and beads not coated with any oligoes served as negative controls in the capture experiments. Full-length blots are presented in Supplementary Fig. S9. C FLAG-HuD RNA binding domain mutants. D FLAG-HuD RNA binding point mutation variants expression by Western blot. Full-length blots are presented in Supplementary Fig. S9. E GRB-10 and ARL6IP1 RNA binding for HuD RBDs; RNAs interact with HuD and require RNA binding capable RRM domains, mainly through RRM1 and RRM2. F HuD-GRB-10 interaction by RIP/RT-qPCR in SK-N-DZ and SK-N-SH cells (IgG vs. anti-HuD). G HuD-ARL6IP1 interaction by RIP/RT-qPCR in SK-N-DZ and SK-N-SH cells (IgG vs. anti-HuD). Data are presented as mean ± SEM; t-test: t-test: *p < 0.05, **p < 0.01, ***p < 0.001 ($$p < 0.01, $$$p < 0.001 in F)