Fig. 3: HuB enrolls DHX9 into translation machinery.

A Confirmation of the enhanced association of HuB with the proteins revealed by proteomic mass spectrometry in response to inflammation stimulation. HEK293 cells were transfected with plasmid expressing GFP or GFP-HuB, and then mock-treated or exposed to TNFα (10 ng/mL) for 1 h. The cytosolic lysates were collected for protein immunoprecipitation assay with GFP antibody and then subjected to western blotting to detect the interaction of GFP-HuB with DHX9, eIF4H as well as RPS3. B, C HuB facilitates the recruitment of DHX9, HuR and the associated mRNAs into polysome fractions. HEK293 cells were transfected with siRNA targeting HuB or control, and incubated with TNFα (10 ng/mL) for 1 h. Then the isolation of ribosomal fractions was undertaken, and the location of HuB, DHX9 and HuR in polysome fraction was analyzed by western blotting (B). Real-time PCR was performed to detect the mRNA levels of TNFα (C). Quantification shows mean ± SD based on three independent experiments, with significance of the difference determined by one-way ANOVA test (A, C). n = 3 independent experiments, **p < 0.01, *p < 0.05. Source data are available in supplementary data 1 for this figure.